LitCovid-sentences  

PMC:7252096 / 73349-126093 JSONTXT 11 Projects

Annnotations TAB TSV DIC JSON TextAE Lectin_function

Id Subject Object Predicate Lexical cue
T464 0-12 Sentence denotes STAR★Methods
T465 14-33 Sentence denotes Key Resources Table
T466 34-71 Sentence denotes REAGENT or RESOURCE SOURCE IDENTIFIER
T467 72-90 Sentence denotes Biological Samples
T468 91-288 Sentence denotes M. mulatta lung, bone marrow, brain, colon, ileum, jejunum, liver, lung, peripheral blood, spleen, thymus, tonsil, and lymph nodes from various sites Washington National Primate Research Center N/A
T469 289-373 Sentence denotes Human lung tissue from surgical excess University of KwaZulu-Natal IRB Code:BE024/09
T470 374-538 Sentence denotes Human non-inflamed ileal pinch biopsies Multi-center clinical study, approved by the Institutional Review Board at Boston Children’s Hospital IRB Code:IRB-P00030890
T471 539-604 Sentence denotes Human nasal lavage University of Massachusetts Medical School N/A
T472 605-715 Sentence denotes Human nasal scraping, polyp scrapings, ethmoid sinus surgical tissue samples Partners HealthCare Institute N/A
T473 716-809 Sentence denotes M. fascicularis lung and granulomatous tissue University of Pittsburgh School of Medicine N/A
T474 810-820 Sentence denotes Antibodies
T475 821-876 Sentence denotes anti-ACE2 human antibody, goat polyclonal R&D Cat#AF933
T476 877-904 Sentence denotes Bacterial and Virus Strains
T477 905-934 Sentence denotes MHV-68 Adler et al., 2000 N/A
T478 935-1009 Sentence denotes Mycobacterium Tuberculosis, Modified Erdman Strain Martin et al., 2017 N/A
T479 1010-1055 Sentence denotes Chemicals, Peptides, and Recombinant Proteins
T480 1056-1094 Sentence denotes 2-Mercaptoethanol Sigma Cat#M3148-25ML
T481 1095-1122 Sentence denotes RLT Buffer QIAGEN Cat#79216
T482 1123-1158 Sentence denotes dNTP New England BioLabs Cat#N0447L
T483 1159-1203 Sentence denotes RNase Inhibitor Fisher Scientific Cat#AM2696
T484 1204-1262 Sentence denotes Maxima RNaseH-minus RT Enzyme Fisher Scientific Cat#EP0753
T485 1263-1290 Sentence denotes MgCl2 Sigma Cat#63069-100ML
T486 1291-1318 Sentence denotes Betaine Sigma Cat#B0300-5VL
T487 1319-1379 Sentence denotes AMPure RNAClean XP RNA-SPRI beads Beckman Coulter Cat#A63987
T488 1380-1427 Sentence denotes AMPure XP SPRI beads Beckman Coulter Cat#A63881
T489 1428-1468 Sentence denotes Guanidinium thiocyanate Sigma Cat#AM9422
T490 1469-1493 Sentence denotes Sarkosyl Sigma Cat#L7414
T491 1494-1538 Sentence denotes Exonuclease I New England BioLabs Cat#M0293S
T492 1539-1585 Sentence denotes Klenow Fragment New England BioLabs Cat#M0212L
T493 1586-1615 Sentence denotes DNase I Roche Cat#10104159001
T494 1616-1661 Sentence denotes Collagenase IV Life Technologies Cat#17104019
T495 1662-1697 Sentence denotes Collagenase D Roche Cat#11088858001
T496 1698-1730 Sentence denotes Liberase TM Roche Cat#5401119001
T497 1731-1764 Sentence denotes TrypLE Thermo Fisher Cat#12604013
T498 1765-1802 Sentence denotes ACK Buffer Thermo Fisher Cat#A1049201
T499 1803-1829 Sentence denotes IFN-α Biolegend Cat#752802
T500 1830-1867 Sentence denotes Dispase II Thermo Fisher Cat#17105041
T501 1868-1909 Sentence denotes Elastase Worthington Biochem Cat#LS002292
T502 1910-1968 Sentence denotes Pneumacult-Ex serum-free media StemCell Technologies, Inc.
T503 1969-1978 Sentence denotes Cat#05040
T504 1979-2011 Sentence denotes IL-4, human Biolegend Cat#574002
T505 2012-2045 Sentence denotes IL17A, human Biolegend Cat#570502
T506 2046-2078 Sentence denotes IFNγ, human Biolegend Cat#570202
T507 2079-2111 Sentence denotes IFNγ, mouse Peprotech Cat#315-05
T508 2112-2144 Sentence denotes IFNα, human Biolegend Cat#592702
T509 2145-2177 Sentence denotes IFNα, mouse Biolegend Cat#752802
T510 2178-2217 Sentence denotes IFNβ, mouse R&D Systems Cat#8234-MB-010
T511 2218-2244 Sentence denotes Critical Commercial Assays
T512 2245-2308 Sentence denotes Nextera XT DNA Library Preparation Kit Illumina Cat#FC-131-1096
T513 2309-2364 Sentence denotes High Sensitivity D5000 ScreenTape Agilent Cat#5067-5592
T514 2365-2421 Sentence denotes Qubit dsDNA High-Sensitivity kit ThermoFisher Cat#Q32854
T515 2422-2489 Sentence denotes NextSeq 500/550 High Output v2 (75 cycles) Illumina Cat#FC-404-2005
T516 2490-2540 Sentence denotes NovaSeq 6000 S2 (100 cycles) Illumina Cat#20012862
T517 2541-2595 Sentence denotes Kapa HiFi HotStart ReadyMix Kapa Biosystems Cat#KK2602
T518 2596-2654 Sentence denotes MACOSKO-2011-10 mRNA Capture Beads ChemGenes Cat#NC0927472
T519 2655-2716 Sentence denotes Tumor Dissociation Kit, Human Miltenyi Biotec Cat#130-095-929
T520 2717-2767 Sentence denotes Chromium Single Cell 3′ v2 10X Genomics Cat#120237
T521 2768-2782 Sentence denotes Deposited Data
T522 2783-2897 Sentence denotes scRNA-seq Processed Data This paper https://singlecell.broadinstitute.org/single_cell?scpbr=the-alexandria-project
T523 2898-3018 Sentence denotes scRNA-seq Processed Data This paper https://drive.google.com/drive/folders/1bxCIqNeZ7wLuVOT16gphwj98_cc9KhrV?usp=sharing
T524 3019-3128 Sentence denotes scRNA-seq Processed Data This paper https://chanzuckerberg.github.io/cellxgene/posts/cellxgene_cziscience_com
T525 3129-3222 Sentence denotes scRNA-seq Processed Data This paper https://singlecell.broadinstitute.org/single_cell/covid19
T526 3223-3333 Sentence denotes scRNA-seq Processed Data (all species) and FASTQ files (for NHP and murine datasets) This paper GEO: GSE148829
T527 3334-3543 Sentence denotes scRNA-seq data from human nasal mucosa Ordovas-Montanes et al., 2018 https://singlecell.broadinstitute.org/single_cell/study/SCP253/allergic-inflammatory-memory-in-human-respiratory-epithelial-progenitor-cells
T528 3544-3681 Sentence denotes Human reference genome NCBI build 38 (GRCh38) Genome Reference Consortium http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/human/
T529 3682-3810 Sentence denotes Human reference genome NCBI build 19 Genome Reference Consortium http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/human/
T530 3811-3939 Sentence denotes Mouse reference genome NCBI build 10 Genome Reference Consortium http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/mouse/
T531 3940-4116 Sentence denotes Macaca mulatta reference genome assembly 8.0.1, annotation 102 NCBI Eukaryotic Genome Annotation Pipeline https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Macaca_mulatta/102/
T532 4117-4299 Sentence denotes Macaca fascicularis reference genome assembly 5, annotation 101 NCBI Eukaryotic Genome Annotation Pipeline https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Macaca_fascicularis/101/
T533 4300-4370 Sentence denotes Interferome Database Rusinova et al., 2013 http://www.interferome.org/
T534 4371-4461 Sentence denotes RNA-seq from human lung explants ± ex vivo IAV infection Matos et al., 2019 GEO: GSE135069
T535 4462-4557 Sentence denotes RNA-seq from human nasal epithelial cells Giovannini-Chami et al., 2012 GEO: GSE19190, GSE22147
T536 4558-4578 Sentence denotes Experimental Models:
T537 4579-4589 Sentence denotes Cell Lines
T538 4590-4596 Sentence denotes Human:
T539 4597-4628 Sentence denotes Passage 4 BEAS-2B ATCC CRL-9609
T540 4629-4649 Sentence denotes Experimental Models:
T541 4650-4667 Sentence denotes Organisms/Strains
T542 4668-4717 Sentence denotes Mouse: C57BL/6J The Jackson Laboratory Cat#000664
T543 4718-4800 Sentence denotes Mouse: C57BL/6, IFNγR−/− B6.129S7-Ifngr1tm1Agt/J The Jackson Laboratory Cat#003288
T544 4801-4817 Sentence denotes Oligonucleotides
T545 4818-4966 Sentence denotes SMART-seq2 2 3′ Oligo-dT Primer: /5Biosg/AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TVN Integrated DNA Technologies N/A
T546 4967-5060 Sentence denotes SMART-seq2 5′ TSO: AAG CAG TGG TAT CAA CGC AGA GTA CAT rGrGrG Integrated DNA Technologies N/A
T547 5061-5153 Sentence denotes SMART-seq2 and Seq-Well ISPCR:AAG CAG TGG TAT CAA CGC AGA GT Integrated DNA Technologies N/A
T548 5154-5257 Sentence denotes Custom Read 1 Primer: GCC TGT CCG CGG AAG CAG TGG TAT CAA CGC AGA GTA C Integrated DNA Technologies N/A
T549 5258-5349 Sentence denotes Seq-Well 5′ TSO: AAG CAG TGG TAT CAA CGC AGA GTG AAT rGrGrG Integrated DNA Technologies N/A
T550 5350-5512 Sentence denotes Seq-Well Custom P5-SMART PCR hybrid oligo: AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG CCT GTC CGC GGA AGC AGT GGT ATC AAC GCA GAG TAC Integrated DNA Technologies N/A
T551 5513-5614 Sentence denotes Seq-Well dN-SMRT oligo: AAG CAG TGG TAT CAA CGC AGA GTG ANN NGG NNN B Integrated DNA Technologies N/A
T552 5615-5638 Sentence denotes Software and Algorithms
T553 5639-5678 Sentence denotes R R Core Team https://www.r-project.org
T554 5679-5758 Sentence denotes R package – Seurat v2.3.4 and v3.1.0 Github https://github.com/satijalab/seurat
T555 5759-5818 Sentence denotes Scanpy Wolf et al., 2018 https://github.com/theislab/scanpy
T556 5819-5887 Sentence denotes R package – SCDE Bioconductor http://bioconductor.org/packages/scde/
T557 5888-5965 Sentence denotes Prism 6 GraphPad Software https://www.graphpad.com/scientific-software/prism/
T558 5966-6011 Sentence denotes STAR Github https://github.com/alexdobin/STAR
T559 6012-6097 Sentence denotes Uniform Manifold Approximation and Projection Github https://github.com/lmcinnes/umap
T560 6098-6155 Sentence denotes Rtsne CRAN https://cran.r-project.org/web/packages/Rtsne/
T561 6157-6178 Sentence denotes Resource Availability
T562 6180-6192 Sentence denotes Lead Contact
T563 6193-6372 Sentence denotes Further information and requests for resources and reagents should be directed to and will be fulfilled by Dr. Jose Ordovas-Montanes (jose.ordovas-montanes@childrens.harvard.edu).
T564 6374-6396 Sentence denotes Materials Availability
T565 6397-6445 Sentence denotes This study did not generate new unique reagents.
T566 6447-6473 Sentence denotes Data and Code Availability
T567 6474-6715 Sentence denotes In Table S9 , we provide a guide to all datasets analyzed in this paper as well as links to each individual dataset for download with the main landing page here: https://singlecell.broadinstitute.org/single_cell?scpbr=the-alexandria-project.
T568 6716-6920 Sentence denotes To download the data from the portal, follow the link to the visualization page, sign in a free account in the portal using a Google apps enabled email address, and select the ‘Download’ tab in the study.
T569 6921-7026 Sentence denotes Downloadable datasets include both raw and normalized cell x gene matrices, as well as relevant metadata.
T570 7027-7186 Sentence denotes These datasets are additionally available here to facilitate downloading: https://drive.google.com/drive/folders/1bxCIqNeZ7wLuVOT16gphwj98_cc9KhrV?usp=sharing.
T571 7187-7491 Sentence denotes We have also posted these cell x gene matrices to Chan Zuckerberg Initiative cellxgene (https://chanzuckerberg.github.io/cellxgene/posts/cellxgene_cziscience_com) and the Broad Institute Single Cell COVID-19 portal (https://singlecell.broadinstitute.org/single_cell/covid19) as leading community efforts.
T572 7492-7635 Sentence denotes FASTQ files and cell x gene matrices for NHP and murine datasets, and cell x gene matrices for human datasets, are available at GEO: GSE148829.
T573 7636-7695 Sentence denotes In this same table, we further highlight four access types.
T574 7696-8183 Sentence denotes 1. published datasets where everything is available (1 study); 2. unpublished datasets where everything is available (2 studies, 19,670 new cells for download), 3. unpublished datasets where ACE2+ cell subsets, and the necessary subsets to contextualize those cells (i.e., epithelial cells for type II pneumocytes) are fully available (5 studies, 17,986 new cells for download); and, 4. those unpublished datasets where expression is shared for ACE2/TMPRSS2 (2 studies, 9,112 new cells).
T575 8184-8359 Sentence denotes For those unpublished datasets where only specific subsets of cells or genes are available, full expression matrices are available upon request for COVID-19 related questions.
T576 8360-8448 Sentence denotes All data included in the present study can be visualized using the following web viewer:
T577 8449-8528 Sentence denotes https://singlecell.broadinstitute.org/single_cell?scpbr=the-alexandria-project.
T578 8529-8898 Sentence denotes As we gain further insight and feedback from our own groups, collaborators, and investigators, we will continue to provide updates on our resource websites, including the utility of in vitro systems, such as organoids (Mead et al., 2018), for the study of SARS-CoV-2: http://shaleklab.com/resource/covid-19-resources/ and www.ordovasmontaneslab.com/covid-19-resources/.
T579 8899-9088 Sentence denotes We also note that there are several ongoing efforts unified together through the HCA Lung Biological Network group that we will reference and to which we will link as they become available.
T580 9089-9209 Sentence denotes No custom code was used to analyze these data and all methods and packages used are cited in the Method Details section.
T581 9211-9249 Sentence denotes Experimental Model and Subject Details
T582 9251-9276 Sentence denotes Human Intestinal Biopsies
T583 9277-9509 Sentence denotes For human intestinal biopsies from the terminal ileum, the subjects were enrolled on a multi-center clinical study, which was approved by the Institutional Review Board at Boston Children’s Hospital (protocol number: IRB-P00030890).
T584 9510-9634 Sentence denotes Full information related to subject age/developmental stage and sex found in metadata associated with provided raw datasets.
T585 9636-9664 Sentence denotes Human Lungs, Surgical Excess
T586 9665-9893 Sentence denotes Samples were obtained through indicated lung lobe resection or diagnostic procedures in collaboration with clinicians at the Department of Cardiothoracic Surgery at Inkosi Albert Luthuli Central Hospital in Durban, South Africa.
T587 9894-9946 Sentence denotes Informed consent was obtained from each participant.
T588 9947-10061 Sentence denotes The study protocol was approved by the University of KwaZulu-Natal Institutional Review Board (approval BE024/09).
T589 10062-10186 Sentence denotes Full information related to subject age/developmental stage and sex found in metadata associated with provided raw datasets.
T590 10188-10220 Sentence denotes Human Nasal Polyps and Scrapings
T591 10221-10499 Sentence denotes For inferior turbinate nasal scrapings, polyp scrapings, and ethmoid sinus surgical tissue samples, the Partners HealthCare Institutional Review Board (Boston, Massachusetts), approved the study and all subjects provided written informed consent (Ordovas-Montanes et al., 2018).
T592 10500-10624 Sentence denotes Full information related to subject age/developmental stage and sex found in metadata associated with provided raw datasets.
T593 10626-10676 Sentence denotes Human Nasal Washes, Healthy and Influenza Infected
T594 10677-10855 Sentence denotes The Institutional Review Board of the University of Massachusetts Medical School (Worcester, Massachusetts) approved the study and all subjects provided written informed consent.
T595 10857-10907 Sentence denotes Cell Culture of Primary Basal Cells and Cell Lines
T596 10908-11282 Sentence denotes Human basal cells from non-polyp surgical resections from ethmoid sinus, BEAS-2B cells (ATCC), or mouse tracheal basal cells were placed into culture at a number of 10,000 cells seeded at passage 3 and cultured at confluence in 96 well flat-bottom collagen-coated tissue culture plates (Corning 3596) for 48 h in Pneumacult-Ex serum-free media (StemCell Technologies, Inc.).
T597 11283-11327 Sentence denotes All cells were incubated at 37°C and 5% CO2.
T598 11329-11360 Sentence denotes Non-Human Primates (M. mulatta)
T599 11361-11583 Sentence denotes Healthy and SHIV-infected non-human primate (M. mulatta) work was conducted at the Washington National Primate Research Center (WaNPRC), an AAALAC accredited program, in accordance with the regulations detailed in the U.S.
T600 11584-11726 Sentence denotes Department of Agriculture Animal Welfare Act and in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
T601 11727-11815 Sentence denotes It was approved by University of Washington Institutional Animal Care and Use Committee.
T602 11816-11892 Sentence denotes Expanded cohort characteristics described previously (Colonna et al., 2018).
T603 11893-12017 Sentence denotes Full information related to subject age/developmental stage and sex found in metadata associated with provided raw datasets.
T604 12019-12055 Sentence denotes Non-Human Primates (M. fascicularis)
T605 12056-12294 Sentence denotes Tissues from Mycobacterium tuberculosis-infected non-human primates (M. fascicularis) were conducted at the University of Pittsburgh School of Medicine, an AAALAC accredited program, in accordance with the regulations detailed in the U.S.
T606 12295-12437 Sentence denotes Department of Agriculture Animal Welfare Act and in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
T607 12438-12562 Sentence denotes Full information related to subject age/developmental stage and sex found in metadata associated with provided raw datasets.
T608 12564-12619 Sentence denotes Mouse Nasal and Olfactory Epithelium and Tracheal Cells
T609 12620-12822 Sentence denotes C57BL/6J mice purchased from Jackson laboratory (Bar Harbor, ME, USA) were maintained within Ragon Institute’s HPPF barrier facility and all experiments were conducted with institutional IACUC approval.
T610 12823-12904 Sentence denotes In this study, mice were 8-10 weeks of age, representing male and female animals.
T611 12906-12934 Sentence denotes Mouse Lungs, MHV68 Infection
T612 12935-13015 Sentence denotes C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany).
T613 13016-13263 Sentence denotes IFNγR−/− mice on C57BL/6 background (C57BL/6, IFNγR−/− B6.129S7-Ifngr1 tm1Agt/J) were originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and subsequently bred and propagated under SPF conditions at the Helmholtz Zentrum München.
T614 13264-13430 Sentence denotes Animals with different genotypes were kept in the same animal room for the time of the experiment including an adaptation period prior to the start of the experiment.
T615 13431-13535 Sentence denotes All animal experiments were in compliance with the German Animal Welfare Act (German Federal Law §8 Abs.
T616 13536-13624 Sentence denotes 1 TierSchG), and the protocols were approved by the local Animal Care and Use Committee.
T617 13626-13640 Sentence denotes Method Details
T618 13642-13717 Sentence denotes Methods of Sample Collection and Tissue Preparation for Single-Cell RNA-Seq
T619 13719-13784 Sentence denotes NHP Ileum, Jejunum, Colon, Liver, Tonsil, Thymus, and Lung Tissue
T620 13785-13948 Sentence denotes Animals were perfused with 0.5 L of PBS/kg immediately following euthanasia, tissues were isolated and placed in RPMI + 10% FBS and kept on ice until dissociation.
T621 13949-14096 Sentence denotes Tissue sections were digested by mincing and incubating with collagenase IV (Life Technologies) and DNase I (Roche) at 37°C for 1 h with agitation.
T622 14097-14327 Sentence denotes Digested tissue was passed through a 100 μm metal strainer, cells were pelleted by centrifugation at 300 g, rinsed with RPMI + 10% FBS, counted, and prepared as a single cell suspension for scRNA-seq using Seq-Well v1 (see below).
T623 14329-14368 Sentence denotes NHP Lymphoid Organs, Bone Marrow, PBMCs
T624 14369-14541 Sentence denotes All lymph nodes, spleen, and bone marrow were ground through a metal strainer, transferred to a conical in RPMI + 10% FBS, and pelleted by centrifugation at 400 g x 10 min.
T625 14542-14644 Sentence denotes LN-derived cells were resuspended in RPMI + 10% FBS, counted and prepared as a single cell suspension.
T626 14645-14765 Sentence denotes Spleen, bone marrow, and PBMCs were subjected to ACK lysis for 10 min at room temperature, quenched with RPMI + 10% FBS.
T627 14766-14925 Sentence denotes PBMCs and bone marrow derived cells were purified over a ficoll gradient (GE Healthcare) by centrifuging at 400 g for 20 min at room temperature with no brake.
T628 14926-15038 Sentence denotes Cells were then resuspended in RPMI + 10% FBS, counted, and diluted for scRNA-seq using Seq-Well v1 (see below).
T629 15040-15084 Sentence denotes NHP Tuberculosis Infected Lung and Granuloma
T630 15085-15218 Sentence denotes Ten Mycobacterium tuberculosis infected (Martin et al., 2017) adult non-human primates (M. fascicularis) were included in this study.
T631 15219-15461 Sentence denotes A piece of lung tissue (without any grossly visible pathology) and 4 individual TB lung granulomas per animal were excised at necropsy and enzymatically dissociated using the GentleMacs system (Tumor dissociation kit, human; Miltenyi Biotec).
T632 15462-15586 Sentence denotes Single cell suspensions were resuspended in RPMI + 10% FBS, counted and diluted for scRNA-seq using Seq-Well S3 (see below).
T633 15588-15605 Sentence denotes Human Lung Tissue
T634 15606-15758 Sentence denotes Surgical samples from diseased lung tissue (n = 3 TB+HIV+; n = 3 TB+; n = 2 non-infected patients) were processed as described in (Ardain et al., 2019).
T635 15759-16028 Sentence denotes Briefly, each sample was collected into cold RP-10 (RPMI (Sigma-Aldrich) + 10% FBS), minced, and incubated for 25-30 min at 37°C with digestion buffer containing collagenase D (Sigma-Aldrich), DNase I (Sigma-Aldrich) in RPMI 1640 (Sigma-Aldrich) with 10% FBS (Hyclone).
T636 16029-16185 Sentence denotes Following incubation, samples were homogenized using a GentleMACS, filtered using a 70 μm metal strainer, and pelleted by centrifugation at 400 g for 5 min.
T637 16186-16394 Sentence denotes After obtaining the pellet, cells were resuspended in RP-10, passed through another 70μm strainer (Corning), stained with trypan blue, and then counted and diluted for scRNA-seq using Seq-Well S3 (see below).
T638 16396-16407 Sentence denotes Human Ileum
T639 16408-16497 Sentence denotes Single-cell suspensions were collected from biopsies as described (Smillie et al., 2019).
T640 16498-16647 Sentence denotes Briefly, biopsies were rinsed in cold PBS, the epithelial layer was separated from the underlying lamina propria by end over end rotation for 15 min.
T641 16648-16842 Sentence denotes The lamina propria and epithelial fractions were digested separately, using Liberase TM (Roche) and DNase I (Roche) for the lamina propria, and TrypLE (ThermoFisher) for the epithelial fraction.
T642 16843-16975 Sentence denotes Following digestion, cells were pelleted by centrifugation, subjected to ACK lysis for 3 min, and filtered through a 40 μm strainer.
T643 16976-17111 Sentence denotes Following centrifugation, the cells were counted and prepared as a single cell suspension for scRNA-seq using 10X 3′ v2 (10X Genomics).
T644 17113-17145 Sentence denotes Nasal Mucosa and Nasal Scrapings
T645 17146-17287 Sentence denotes Surgical samples from ethmoid sinus and nasal scraping of the inferior turbinate were processed as described (Ordovas-Montanes et al., 2018).
T646 17288-17518 Sentence denotes Briefly, each sample was collected into cold RPMI (Corning), minced and incubated for 30 min (15 min for nasal scrapings) at 37°C with digestion buffer containing collagenase IV (Worthington), DNase I (Roche) in RPMI with 10% FBS.
T647 17519-17576 Sentence denotes Samples were triturated and digestion quenched with EDTA.
T648 17577-17886 Sentence denotes Cells were filtered using a 70 μm metal strainer and pelleted by centrifugation at 500 g, rinsed with PBS, and subjected to red blood cell (RBC) lysis using ACK buffer (ThermoFisher) for 3 min on ice, and finally pelleted prepared as a single cell suspension for scRNA-seq using Seq-Well v1 or S3 (see below).
T649 17888-17930 Sentence denotes Interferon Treatment of Mouse Nasal Mucosa
T650 17931-18064 Sentence denotes Mice received either 200ng of IFNα (Biolegend 752802) or saline intranasally (each group n = 2 mice), and were sacrificed 12 h later.
T651 18065-18164 Sentence denotes Respiratory and olfactory mucosa were isolated as in (Davidson et al., 2004, Dunston et al., 2013).
T652 18165-18307 Sentence denotes Briefly, using surgical tools under a dissecting microscope, the skull bones surrounding the nasal tissue of skinned mouse heads were removed.
T653 18308-18387 Sentence denotes The respiratory and olfactory mucosa were collected in RPMI media with 10% FBS.
T654 18388-18502 Sentence denotes Cells were digested in media containing Liberase TM (Roche) and DNase I (Roche) for 30 min at 37°C with agitation.
T655 18503-18643 Sentence denotes Cells were filtered using a 70 μm strainer, washed with EDTA-containing media to quench enzymatic digestion, and pelleted by centrifugation.
T656 18644-18820 Sentence denotes RBCs were lysed using ACK buffer (ThermoFisher) for 2 min, cells were again pelleted, counted, and prepared as a diluted single cell suspension for scRNA-seq using Seq-Well S3.
T657 18822-18847 Sentence denotes MHV68 Infected Mouse Lung
T658 18848-18932 Sentence denotes Mice were housed in individually ventilated cages during the MHV68 infection period.
T659 18933-19033 Sentence denotes MHV68 stocks were grown and quantified by plaque assay as previously described (Adler et al., 2000).
T660 19034-19159 Sentence denotes Mice were infected intranasally (i.n.) with 5 × 10∗4 plaque forming units of MHV68 diluted in PBS in a total volume of 30 μl.
T661 19160-19245 Sentence denotes Prior to i.n. infection, mice were anesthetized with medetomidine–midazolam–fentanyl.
T662 19246-19382 Sentence denotes At the predetermined time points, mice were sacrificed by cervical dislocation and lung tissue was processed for subsequent experiments.
T663 19383-19610 Sentence denotes All lobes were removed, minced and transferred for mild enzymatic digestion for 20-30 min at 37°C in an enzymatic mix containing Dispase (50 caseinolytic U/mL), Collagenase (2 mg/mL), Elastase (1 mg/mL), and DNase I (30 μg/mL).
T664 19611-19707 Sentence denotes Single cells were harvested by straining the digested tissue suspension through a 70μm strainer.
T665 19708-19819 Sentence denotes After centrifugation at 300 x g for 5 min, single cells were counted, and prepared as a single cell suspension.
T666 19820-19951 Sentence denotes For Drop-seq, cells were aliquoted in PBS supplemented with 0.04% of bovine serum albumin at a final concentration of 100 cells/μl.
T667 19953-19992 Sentence denotes Nasal Washes during Influenza Infection
T668 19993-20507 Sentence denotes Nasal washes were obtained from adult healthy controls and from adults with diagnosis of acute influenza A or B by rapid antigen test (Flu A or B antigen, direct fluorescence antigen test) and/or by respiratory virus panel (PCR testing for influenza A, influenza A H1, influenza A H3, influenza B, adenovirus, metapneumovirus, respiratory syncytial virus A, respiratory syncytial virus B, rhino/enterovirus, parainfluenza 1, parainfluenza 2, parainfluenza 3), who show symptoms up to seven days (Cao et al., 2020).
T669 20508-20622 Sentence denotes Samples were obtained by irrigation of each naris with up to 10 mL of saline, and collected in a single container.
T670 20623-20697 Sentence denotes The sample was then transported to the research laboratory for processing.
T671 20698-20838 Sentence denotes Upon receipt, the sample was immediately stored on ice and 10 mL cell growth media (DMEM or RPMI1640 with 10% fetal bovine serum) was added.
T672 20839-20938 Sentence denotes The material was strained using a 40 μm nylon cell strainer (Corning) into a 50 mL centrifuge tube.
T673 20939-20989 Sentence denotes Cells were pelleted at 1300 rpm for 10 min at 4°C.
T674 20990-21179 Sentence denotes All but 1 mL of supernatant was discarded, the pellet resuspended in the remaining 1 mL of supernatant, and material was transferred to an Eppendorf tube and pelleted at 2000 rpm for 5 min.
T675 21180-21447 Sentence denotes If the pellet contained visible blood, 200 μL of RBC lysis solution (ACK buffer, Thermo Fisher) was added to resuspend the pellet and incubated at room temperature for 2 min, after which 1 mL of cell media was added, and the cells were pelleted at 2000 rpm for 5 min.
T676 21448-21564 Sentence denotes The final pellet was resuspended in up to 1 mL of media and quantified before performing scRNA-seq with Seq-Well v1.
T677 21566-21624 Sentence denotes Methods to Generate Single-Cell and Bulk RNA-seq Libraries
T678 21626-21637 Sentence denotes Seq-Well v1
T679 21638-21697 Sentence denotes Seq-Well was performed as described (Gierahn et al., 2017).
T680 21698-21816 Sentence denotes Single cells were diluted to 15,000 cells in 200 μL RPMI + 10% FBS and deposited onto a pre-functionalized PDMS array.
T681 21817-21923 Sentence denotes 15,000 cells were deposited onto the top of each PDMS array and let settle by gravity into distinct wells.
T682 21924-22019 Sentence denotes The array was gently washed with PBS, and sealed using a functionalized polycarbonate membrane.
T683 22020-22235 Sentence denotes Seq-Well arrays were sealed in a dry 37°C oven for 40 min, and submerged in a lysis buffer containing guanidium thiocyanate (Sigma), EDTA, 1% beta-mercaptoethanol and sarkosyl (Sigma) for 20 min at room temperature.
T684 22236-22726 Sentence denotes Arrays were transferred to hybridization buffer containing NaCl (Fisher Scientific) and agitated for 40 min at room temperature, mRNA capture beads with mRNA hybridized were collected from each Seq-Well array, and beads were resuspended in a master mix for reverse transcription containing Maxima H Minus Reverse Transcriptase and buffer, dNTPs, RNase inhibitor, a 5′ template switch oligonucleotide, and PEG for 30 min at room temperature, and overnight at 52°C with end-over-end rotation.
T685 22727-22787 Sentence denotes Exonuclease digestion and PCR were carried out as described.
T686 22788-22922 Sentence denotes Post-whole transcriptome amplification workup involved AMPure XP SPRI bead cleanup occurred at a 0.6 x volume ratio, followed by 0.8x.
T687 22923-23086 Sentence denotes Library size was analyzed using an Agilent Tapestation hsD5000 kit, confirming the expected peak at ∼1000 bp, and absence of smaller peaks corresponding to primer.
T688 23087-23295 Sentence denotes Libraries were quantified using Qubit High-Sensitivity DNA kit and prepared for Illumina sequencing using Nextera XT DNA Sample Preparation kit using 900 pg of cDNA library as input to tagmentation reactions.
T689 23296-23424 Sentence denotes Amplified final libraries were purified twice with AMPure XP SPRI beads as before, with a volume ratio of 0.6x followed by 0.8x.
T690 23425-23618 Sentence denotes Libraries from 2-3 Seq-Well arrays were pooled and sequenced together using a NextSeq 500/550 High Output v2 kit (75 cycles) using a paired end read structure with custom read 1 primer: read 1:
T691 23619-23636 Sentence denotes 20 bases, read 2:
T692 23637-23660 Sentence denotes 50 bases, read 1 index:
T693 23661-23669 Sentence denotes 8 bases.
T694 23671-23682 Sentence denotes Seq-Well S3
T695 23683-23770 Sentence denotes Seq-Well S3 modified the following protocol steps from v1, above (Hughes et al., 2019).
T696 23771-23861 Sentence denotes First, hybridization buffer was supplanted with 8% (v/v) polyethylene glycol (PEG, Sigma).
T697 23862-23986 Sentence denotes Second, after exonuclease digestion, bead-associated cDNA was denatured for 5 min in 0.2 mM NaOH with end over end rotation.
T698 23987-24239 Sentence denotes Next, beads were washed with TE + 0.01% tween-20, and second strand synthesis was carried out by resuspending beads in a master mix containing Klenow Fragment (NEB), dNTPs, PEG, and the dN-SMRT oligonucleotide to enable random priming off of the beads.
T699 24241-24250 Sentence denotes 10X v2 3′
T700 24251-24388 Sentence denotes Single cells were loaded onto 3′ library chips as per the manufacturers protocol for Chromium Single Cell 3′ Library (v2) (10X Genomics).
T701 24389-24653 Sentence denotes Each biopsy was sequenced on two channels of the 10X Chromium Single Cell Platform, one for the epithelial fraction and the other for the lamina propria fraction in order to recover sufficient numbers of epithelial and lamina propria cells for downstream analyses.
T702 24654-24761 Sentence denotes An input of 6,000 single cells was added to each channel with a recovery rate of approximately 2,000 cells.
T703 24763-24771 Sentence denotes Drop-seq
T704 24772-24866 Sentence denotes Drop-seq experiments were performed according to the original protocol (Macosko et al., 2015).
T705 24867-24993 Sentence denotes Briefly, single cells (100/μl) were co-encapsulated in droplets with barcoded beads (120/μl, ChemGenes) at rates of 4000 μl/h.
T706 24994-25108 Sentence denotes Droplet emulsions were collected for 10-20 min/each prior to droplet breakage by perfluorooctanol (Sigma-Aldrich).
T707 25109-25229 Sentence denotes After breakage, beads were harvested and the hybridized mRNA transcripts reverse transcribed (Maxima RT, Thermo Fisher).
T708 25230-25306 Sentence denotes Exonuclease digestion and PCR were carried out as described (12 PCR cycles).
T709 25307-25473 Sentence denotes For each sample, 1 ng of pre-amplified cDNA from an estimated 1000 cells was tagmented by Nextera XT (Illumina) with a custom P5-primer (Integrated DNA Technologies).
T710 25474-25564 Sentence denotes Single-cell libraries were sequenced in a 100 bp paired-end run on the Illumina HiSeq4000.
T711 25566-25593 Sentence denotes Smart-Seq2 for Bulk RNA-Seq
T712 25594-25696 Sentence denotes Population RNA-seq was performed as described (Ordovas-Montanes et al., 2018, Trombetta et al., 2014).
T713 25697-25993 Sentence denotes Briefly, RNA from population lysates was purified using AMPure RNA Clean Spri beads (Beckman Coulter) at a 2.2x volume ratio, and mixed with oligo-dT primer, dNTPs (NEB), and RNase inhibitor (Fisher Scientific) at 72°C for 3 min on a thermal cycler to anneal the 3′ primer to polyadenylated mRNA.
T714 25994-26326 Sentence denotes Reverse transcription was carried out in a master mix of Maxima RNaseH-minus RT enzyme and buffer (Fisher Scientific), MgCl2 (Sigma), Betaine (Sigma), RNase inhibitor, and a 5′ template switch oligonucleotide, and PCR was carried out using KAPA HiFi HotStart ReadyMix (Kapa Biosystems) and IS PCR primer and amplified for 18 cycles.
T715 26327-26421 Sentence denotes Libraries were purified using AMPure XP SPRI beads at a volume ratio of 0.8x followed by 0.9x.
T716 26422-26566 Sentence denotes Library size was assessed using a High-Sensitivity DNA chip (Agilent Bioanalyzer), confirming the expected size distribution of ∼1,000-2,000 bp.
T717 26567-26767 Sentence denotes Tagmentation reactions were carried out with the Nextera XT DNA Sample Preparation Kit (Illumina) using 250 pg of cDNA per single cell as input, with modified manufacturer’s instructions as described.
T718 26768-26982 Sentence denotes Libraries were purified twice with AMPure XP SPRI beads at a volume ratio of 0.9x, size distribution assessed using a High Sensitivity DNA chip (Agilent Bioanalyzer) and Qubit High-Sensitivity DNA kit (Invitrogen).
T719 26983-27141 Sentence denotes Libraries were pooled and sequenced using NextSeq500/550 High Output v2 kits (75 cycles, Illumina) using 30-30 paired end sequencing with 8-mer dual indexing.
T720 27143-27190 Sentence denotes Human and Mouse Basal Cell Cytokine Stimulation
T721 27191-27225 Sentence denotes Data represented in Figures 5A–5L:
T722 27226-27334 Sentence denotes Cytokines were added for 12 h overnight at increasing doses (0, 0.1, 0.5, 1, 2, 5, 10 ng/mL) of IL-4 (human:
T723 27335-27368 Sentence denotes Biolegend 574002), IL-17A (human:
T724 27369-27400 Sentence denotes Biolegend 570502), IFNγ (human:
T725 27401-27425 Sentence denotes Biolegend 570202; mouse:
T726 27426-27457 Sentence denotes Peprotech 315-05), IFNα (human:
T727 27458-27482 Sentence denotes Biolegend 592702; mouse:
T728 27483-27543 Sentence denotes Biolegend 752802), or IFNβ (mouse: R&D Systems 8234-MB-010).
T729 27544-27594 Sentence denotes Each condition was run as a biological triplicate.
T730 27595-27935 Sentence denotes Data represented in Figure S3C-K: cytokines were added for 12 h overnight at increasing doses (0, 0.1, 0.5, 1, 5, 10 ng/mL) of human IL-4 (Biolegend 574004), IL-13 (Biolegend 571104), IFNα (Biolegend 592704), IFNγ (Biolegend 570204), IL-17A (Biolegend 570504), or IL-1β (Biolegend 579404) (each condition run as a biological quadruplicate).
T731 27936-28059 Sentence denotes All populations were lysed in 50 μL lysis buffer (RLT + 1% BME, QIAGEN and Sigma, respectively) and snap frozen on dry ice.
T732 28060-28164 Sentence denotes Bulk RNA-seq was performed as described previously and summarized above (Ordovas-Montanes et al., 2018).
T733 28165-28368 Sentence denotes Populations were sequenced to an average ± SEM read depth of 3.95 ± 0.11 million reads per sample, with an average ± SEM alignment percentage to either hg19 or mm10 reference transcriptomes of 71 ± 0.3%.
T734 28369-28451 Sentence denotes All samples met quality thresholds regarding genomic and transcriptomic alignment.
T735 28453-28480 Sentence denotes Western blot for human ACE2
T736 28481-28624 Sentence denotes Established air-liquid interface cultures from bronchial brushings of four asthmatic patients were treated with 10ng/μL of human IFNγ for 24 h.
T737 28625-28756 Sentence denotes Protein lysates were prepared, and anti-ACE2 human antibody (AF933 R&D goat polyclonal) was used to probe for ACE2 by western blot.
T738 28757-28869 Sentence denotes Bands were normalized to GAPDH as loading control, and fold change was computed based on normalized ACE2 values.
T739 28871-28910 Sentence denotes Quantification and Statistical Analysis
T740 28912-28944 Sentence denotes Non-Human Primate Lung and Ileum
T741 28945-29060 Sentence denotes Libraries corresponding to 7 animals (variable number of tissues per animal) were sequenced using Illumina NextSeq.
T742 29061-29256 Sentence denotes Reads were aligned to the M. mulatta genome assembly 8.0.1 annotation version 102 and processed according to the Drop-Seq Computational Protocol v2.0 (https://github.com/broadinstitute/Drop-seq).
T743 29257-29430 Sentence denotes Data was normalized and scaled using the Seurat R package v2.3.4 (https://satijalab.org/seurat/): transforming the data to loge(UMI+1) and applying a scale factor of 10,000.
T744 29431-29663 Sentence denotes To identify major axes of variation within our data, we first examined only highly variable genes across all cells, yielding approximately 1,000-3,000 variable genes with average expression > 0.1 log-normalized UMI across all cells.
T745 29664-29776 Sentence denotes An approximate principal component analysis was applied to the cells to generate 100 principal components (PCs).
T746 29777-29925 Sentence denotes Using the JackStraw function within Seurat, we identified significant PCs to be used for subsequent clustering and further dimensionality reduction.
T747 29926-30161 Sentence denotes For 2D visualization and cell type clustering, we used a Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction technique (https://github.com/lmcinnes/umap) with “min_dist” set to 0.5 and “n_neighbors” set to 30.
T748 30162-30402 Sentence denotes To identify clusters of transcriptionally similar cells, we employed unsupervised clustering as described above using the FindClusters tool within the Seurat R package with default parameters and k.param set to 10 and resolution set to 0.5.
T749 30403-30688 Sentence denotes Each cluster was sub-clustered to identify more granular cell types, requiring each cell type to express > 25 significantly upregulated genes by differential expression test (FindMarkers implemented in Seurat, setting “test.use” to “bimod,” Bonferroni-adjusted p value cutoff < 0.001).
T750 30689-30869 Sentence denotes Differential expression tests between cells from ACE2 + versus ACE2 - Type II Pneumocytes were conducted using the SCDE R package with default parameters (Kharchenko et al., 2014).
T751 30870-31093 Sentence denotes Expression data for epithelial cells and enterocytes included in this dataset can be visualized and downloaded here: https://singlecell.broadinstitute.org/single_cell/study/SCP807?scpbr=the-alexandria-project#study-summary.
T752 31095-31112 Sentence denotes Human Lung Tissue
T753 31113-31187 Sentence denotes Libraries corresponding to 8 donors were sequenced using Illumina NextSeq.
T754 31188-31348 Sentence denotes Reads were aligned to the hg19 genome assembly and processed according to the Drop-Seq Computational Protocol v2.0 (https://github.com/broadinstitute/Drop-seq).
T755 31349-31522 Sentence denotes Data was normalized and scaled using the Seurat R package v3.1.0 (https://satijalab.org/seurat/), transforming the data to loge(UMI+1) and applying a scale factor of 10,000.
T756 31523-31659 Sentence denotes For each array, we assessed the quality of constructed libraries by examining the distribution of reads, genes and transcripts per cell.
T757 31660-31789 Sentence denotes Variable gene selection, principal components analysis, and selection of significant principal components was performed as above.
T758 31790-31971 Sentence denotes We visualized our results in a two-dimensional space using UMAP (https://github.com/lmcinnes/umap), and annotated each cluster based on the identification of highly expressed genes.
T759 31972-32133 Sentence denotes To further characterize substructure within cell types (for example, T cells), we performed dimensionality reduction (PCA) and clustering over those cells alone.
T760 32134-32429 Sentence denotes Sub-clusters (i.e., clusters within broad cell type classifications) were annotated by cross-referencing cluster-defining genes with curated gene lists and online databases SaVanT (http://newpathways.mcdb.ucla.edu/savant-dev/) and GSEA/MsigDB (https://www.gsea-msigdb.org/gsea/msigdb/index.jsp).
T761 32430-32706 Sentence denotes Proliferating cells from the human lung (Figure 2C) express high levels of mitotic markers, such as MKI67, and represent primarily T cells (CD3D, CD3E), B cells/antibody-secreting cells (IGJ, MZB1, IGHG1), and myeloid cells (CD14, APOE) and represent a composite cell cluster.
T762 32707-32915 Sentence denotes Differential expression analysis between ACE2+ TMPRSS2+ and negative type II pneumocytes was performed in Seurat using a likelihood-ratio test (FindMarkers implemented in Seurat, setting “test.use” to bimod).
T763 32916-33123 Sentence denotes Expression data for epithelial cells included in this dataset can be visualized and downloaded here: https://singlecell.broadinstitute.org/single_cell/study/SCP814?scpbr=the-alexandria-project#study-summary.
T764 33125-33136 Sentence denotes Human Ileum
T765 33137-33310 Sentence denotes Libraries corresponding to 13 donors were sequenced using Illumina NovaSeq S2 with a Read 1 26bp, Read 2 91bp, Index 1 8bp configuration before reads were aligned to GRCh38.
T766 33311-33557 Sentence denotes Each sample was filtered individually for low quality cells and genes by analyzing distributions of reads, transcripts, percent reads mapped to mitochondrial genes, and complexity per cell, then merged as an outer join to create a single dataset.
T767 33558-33670 Sentence denotes Clustering and differential expression tests were processed using Seurat v3.1.0 (https://satijalab.org/seurat/).
T768 33671-33788 Sentence denotes Normalization and variable gene selection was processed with SCTransform (https://github.com/ChristophH/sctransform).
T769 33789-33979 Sentence denotes Clustering for major cell types was performed using Louvain clustering on dimensionally reduced PCA space with resolution set via grid search optimizing for maximum average silhouette score.
T770 33980-34105 Sentence denotes Due to the scale of the dataset, a randomized subsampling from across the dataset was used to calculate the silhouette score.
T771 34106-34419 Sentence denotes We annotated clusters based on highly expressed genes, then sub-clusters were characterized by performing PCA dimensionality reduction and clustering over those cells alone, and annotated based on highly expressed genes found via one-versus-rest differential expression test (Wilcoxon) within the major cell type.
T772 34420-34588 Sentence denotes Differential expression analysis between ACE2 + TMPRSS2 + and negative epithelial cells was performed in Seurat using a Wilcoxon test and Bonferroni p value correction.
T773 34589-34796 Sentence denotes Expression data for epithelial cells included in this dataset can be visualized and downloaded here: https://singlecell.broadinstitute.org/single_cell/study/SCP812?scpbr=the-alexandria-project#study-summary.
T774 34798-34822 Sentence denotes Human Adult Nasal Mucosa
T775 34823-34919 Sentence denotes Sample processing, sequencing, and analysis was performed as in (Ordovas-Montanes et al., 2018).
T776 34920-35113 Sentence denotes Briefly, scRNA-seq cell suspensions were freshly processed using Seq-Well v1 and Seurat v2.3.4 was utilized for computational analyses presented here (Butler et al., 2018, Satija et al., 2015).
T777 35114-35306 Sentence denotes Cell by gene matrix and R code for initialization of object available to download as Supplemental Data and Supplementary Tables here https://www.nature.com/articles/s41586-018-0449-8 and here:
T778 35307-35496 Sentence denotes http://shaleklab.com/resource/mapping-allergic-inflammation/? and visualized here: https://singlecell.broadinstitute.org/single_cell/study/SCP253?scpbr=the-alexandria-project#study-summary.
T779 35497-35760 Sentence denotes Scores for various cytokines acting on human airway epithelial cells were calculated based on gene lists derived for (Ordovas-Montanes et al., 2018), calculated using AddModuleScore function Seurat, and effect size calculated by Cohen’s d, as previously reported.
T780 35762-35828 Sentence denotes Granulomatous Tissue from Mycobacterium Tuberculosis Infected NHPs
T781 35829-35944 Sentence denotes Libraries corresponding to 10 animals (variable number of tissues/animal) were sequenced using Illumina NovaSeq S2.
T782 35945-36091 Sentence denotes Data was aligned using the Dropseq-tools pipeline on Terra (app.terra.bio) to M. fascicularis reference genome assembly 5, annotation version 101.
T783 36092-36207 Sentence denotes Clustering was performed using Leiden clustering in the Scanpy (scanpy.readthedocs.io) package (Wolf et al., 2018).
T784 36208-36264 Sentence denotes Cell type labels were assigned using known marker genes.
T785 36265-36411 Sentence denotes In this analysis, we include all epithelial cell subsets (secretory, multiciliated, type II pneumocytes, and type I pneumocytes) from all samples.
T786 36412-36675 Sentence denotes Differential expression between ACE2 + TMPRSS2 + cells and other cells of the matched cell subtype (e.g., Secretory Cells) were performed using the “bimod” likelihood-ratio test within each cell subtype and filtered on Benjamini-Hochberg-corrected p value < 0.05.
T787 36676-36776 Sentence denotes Expression data for epithelial cells included in this dataset can be visualized and downloaded here:
T788 36777-36883 Sentence denotes https://singlecell.broadinstitute.org/single_cell/study/SCP806?scpbr=the-alexandria-project#study-summary.
T789 36885-36916 Sentence denotes Basal Cell Cytokine Stimulation
T790 36917-36998 Sentence denotes Libraries corresponding to 279 populations were sequenced using Illumina NextSeq.
T791 36999-37179 Sentence denotes Reads were aligned to the hg19 or mm10 genome assembly using the cumulus platform https://cumulus-doc.readthedocs.io/en/0.12.0/smart_seq_2.html and output as TPM using RSEM v1.3.2.
T792 37180-37268 Sentence denotes Populations were transformed to transcripts per 10K reads and log2(1+TP10K) transformed.
T793 37269-37405 Sentence denotes ACE2 expression by stimulation condition and dose were assessed using one-way ANOVA with post hoc testing using a Bonferroni correction.
T794 37406-37670 Sentence denotes Plots were generated using ggplot2, and transcriptome-wide differential expression was calculated using the Seurat R package v3.1.0 (https://satijalab.org/seurat), function FindMarkers with test.use = ”bimod.” Expression data can be visualized and downloaded here:
T795 37671-37763 Sentence denotes https://singlecell.broadinstitute.org/single_cell/study/SCP822?scpbr=the-alexandria-project.
T796 37765-37807 Sentence denotes Interferon Treatment of Mouse Nasal Mucosa
T797 37808-37971 Sentence denotes Libraries corresponding to 4 mice, with 2 Seq-Well arrays per mouse were sequenced using Illumina NextSeq as described (Gierahn et al., 2017, Hughes et al., 2019).
T798 37972-38123 Sentence denotes Reads were aligned to the mm10 genome and processed according to the Drop-Seq Computational Protocol v2.0 (https://github.com/broadinstitute/Drop-seq).
T799 38124-38297 Sentence denotes Data was normalized and scaled using the Seurat R package v2.3.4 (https://satijalab.org/seurat/): transforming the data to loge(UMI+1) and applying a scale factor of 10,000.
T800 38298-38364 Sentence denotes Cells with fewer than 1000 UMIs and 500 unique genes were removed.
T801 38365-38525 Sentence denotes To identify major axes of variation within our data, we first examined only highly variable genes across all cells, yielding approximately 5,000 variable genes.
T802 38526-38638 Sentence denotes An approximate principal component analysis was applied to the cells to generate 200 principal components (PCs).
T803 38639-38824 Sentence denotes Using a combination of the Jackstraw function in Seurat and observing the “elbow” of the standard deviations of PCs, we chose the top 70 PCs for subsequent clustering and visualization.
T804 38825-39035 Sentence denotes For 2D visualization, we used a Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction technique (https://github.com/lmcinnes/umap) with “min_dist” set to 0.3 and “n_neighbors” set to 50.
T805 39036-39250 Sentence denotes To identify clusters of transcriptionally similar cells, we employed unsupervised clustering as described above using the FindClusters tool within the Seurat R package with default parameters and k.param set to 10.
T806 39251-39344 Sentence denotes Resolution was chosen based on maximization of the average silhouette width across all cells.
T807 39345-39583 Sentence denotes Clusters were merged if a cell type expressed fewer than 25 significantly upregulated genes by differential expression test (FindAllMarkers implemented in Seurat, setting “test.use” to “bimod,” Bonferroni-adjusted p value cutoff < 0.001).
T808 39584-39744 Sentence denotes Differential expression tests between cells from saline-treated or IFNa-treated mice were assessed using the FindMarkers function with “test.use” set to “bimod.
T809 39745-39796 Sentence denotes This dataset can be visualized and downloaded here:
T810 39797-39903 Sentence denotes https://singlecell.broadinstitute.org/single_cell/study/SCP832?scpbr=the-alexandria-project#study-summary.
T811 39905-39950 Sentence denotes Lung from MHV68-Infected WT and IFNγR KO Mice
T812 39951-40083 Sentence denotes Libraries corresponding to 14 mice were aligned to a custom reference genome encompassing both murine (mm10) and herpes virus genes:
T813 40084-40188 Sentence denotes 84 known genes from MHV68 were retrieved from NCBI (NCBI: txid33708) and added to the mm10 mouse genome.
T814 40189-40348 Sentence denotes Reads were aligned to the custom joint genome and processed according to the Drop-Seq Computational Protocol v2.0 (https://github.com/broadinstitute/Drop-seq).
T815 40349-40493 Sentence denotes Barcodes with < 200 unique genes, > 20,000 UMI counts, and > 30% of transcript counts derived from mitochondrially encoded genes were discarded.
T816 40494-40751 Sentence denotes Data analysis was performed using the Scanpy Package following the common procedure, the expression matrices were normalized using scran’s size factor based approach and log transformed via scanpy’s pp.log1p() function (Lun et al., 2016, Wolf et al., 2018).
T817 40752-40937 Sentence denotes SoupX was utilized to reduce ambient RNA bias, using default parameters with pCut set to 0.3, and was applied to each sample before merging the count matrices (Young and Behjati, 2020).
T818 40938-40985 Sentence denotes UMI per cell and cell cycle were regressed out.
T819 40986-41224 Sentence denotes Highly variable genes were selected by running pp.highly_variable_genes() for each sample separately, returning the top 4,000 variable genes per sample, and genes identified in variable in > 5 samples were retained, yielding 14,305 genes.
T820 41225-41412 Sentence denotes Next, only Epcam+ cells were considered, principal components (PCs) were calculated using only the selected variable genes, and 6 PCs were used to perform unsupervised Louvain clustering.
T821 41413-41564 Sentence denotes Type I Pneumocytes were excluded from this analysis based on uniformly negative expression of Ace2, resulting in a final dataset subset of 5,558 cells.
T822 41565-41639 Sentence denotes Cells were identified as infected if at least one viral read was detected.
T823 41641-41680 Sentence denotes Nasal Washes during Influenza Infection
T824 41681-41764 Sentence denotes Sample processing, sequencing, and analysis was performed as in (Cao et al., 2020).
T825 41765-41847 Sentence denotes Reads were aligned to the GRCh37 reference genome combined with influenza genomes.
T826 41848-42033 Sentence denotes Mapped reads from each sample were then corrected for Drop-seq barcode synthesis error using the Drop-seq core computational tools developed by the McCarroll Lab (Macosko et al., 2015).
T827 42034-42195 Sentence denotes Genes were quantified using End Sequence Analysis Toolkit (ESAT, github/garber-lab/ESAT) with parameters -wlen 100 -wOlap 50 -wExt 0 -scPrep (Derr et al., 2016).
T828 42196-42393 Sentence denotes Finally, UMIs that likely result from sequencing errors were corrected by merging any UMIs that were observed only once and have 1 hamming distance from a UMI detected by two or more aligned reads.
T829 42394-42453 Sentence denotes Only cell barcodes with more than 1,000 UMIs were analyzed.
T830 42454-42522 Sentence denotes Cell barcodes with mostly erythrocyte genes (HBA, HBB) were removed.
T831 42523-42609 Sentence denotes From here on, the remaining cell barcodes in the matrix would be referred to as cells.
T832 42610-42704 Sentence denotes The final gene by cell matrix was normalized using the scran package v3.10 (Lun et al., 2016).
T833 42705-42900 Sentence denotes The normalized matrix was used for dimensionality reduction by first selecting variable genes that had a high coefficient of variance (CV) and were expressed (> = 1 UMI) by more than three cells.
T834 42901-43129 Sentence denotes Influenza viral genes, interferon stimulated genes, and cell cycle related genes were removed from the variable gene list in order to minimize the impact of viral responses and mitosis on clustering and cell type identification.
T835 43130-43340 Sentence denotes This resulted in the selection of 2484 variable genes. t-distributed stochastic neighbor embedding (tSNE) was applied to the first ten principal components (PCs), which explained 95% of the total data variance.
T836 43341-43529 Sentence denotes Density clustering (Rodriguez and Laio, 2014) was performed on the resulting tSNE coordinates and identified four major clusters: epithelial cells, neutrophils, macrophages and leukocytes.
T837 43530-43690 Sentence denotes The epithelial cell cluster and the leukocyte cluster were then re-clustered independently, as described above, to identify populations within each metacluster.
T838 43691-43885 Sentence denotes Specifically, the epithelial cell cluster was re-embedded using 2629 variable genes selected by the same criteria mentioned in the previous section and 13 PCs that explained 95% of the variance.
T839 43886-43959 Sentence denotes Density clustering over the epithelial cell subset revealed ten clusters.
T840 43960-44090 Sentence denotes Differential gene expression analysis using edgeR (Robinson et al., 2010) was performed to identify marker genes for each cluster.
T841 44091-44400 Sentence denotes Influenza-infected and bystander cells were identified after correcting for sample-specific distribution of ambient influenza mRNA contamination and predicted cells most likely to be infected identified using a hurdle zero inflated negative binomial (ZINB) model and a support vector machine (SVM) classifier.
T842 44402-44454 Sentence denotes Power Calculations for Detection of Rare Transcripts
T843 44455-44712 Sentence denotes We conducted the following statistical analysis to estimate the effects of various factors on our ability to make confident claims regarding the presence/absence of transcripts of interest (e.g., ACE2), both within individual cells and clusters (Figure S6).
T844 44713-44856 Sentence denotes Specifically, we investigated the roles of capture/reverse transcription efficiency, ACE2 expression level, sequencing depth, and cell numbers.
T845 44857-45113 Sentence denotes Taken together, the results of this power analysis are in agreement with other efforts to model biological and technical sources of zero-inflation within scRNA-seq data (e.g., https://satijalab.org/howmanycells and Kharchenko et al., 2014, Svensson, 2020).
T846 45114-45313 Sentence denotes We began by quantifying how likely we are to capture and transcribe at least one ACE2 mRNA molecule, as a function of the number ACE2 mRNA molecules per cell and a protocol’s efficiency (Figure S6A).
T847 45314-45680 Sentence denotes Drop-Seq has a capture/transcription efficiency of ∼10% (as estimated using ERCC spike ins; see (Macosko et al., 2015), and the experimental platforms used in this study are either equivalent (e.g., Seq-Well v1, (Gierahn et al., 2017) or superior (e.g., 10-fold better unique molecule detection, 5-fold better gene detection using Seq-Well S3;(Hughes et al., 2019)).
T848 45681-45808 Sentence denotes Most relevant to this context, inferior turbinate scrapings were processed using both Seq-Well v1 and Seq-Well S3 (Figure S3B).
T849 45809-45928 Sentence denotes Importantly, Seq-Well S3 provided > two-fold increase in the detection frequency of rare ACE2 transcripts (i.e., ACE2+:
T850 45929-46137 Sentence denotes 4.7% for v1 versus 9.8% for S3), making it reasonable to expect that such improvements in single-cell experimental technologies have yielded corresponding improvements in capture and transcription efficiency.
T851 46138-46647 Sentence denotes Based on Drop-Seq’s 10% efficiency, even if ACE2 is expressed at the low level of 5 mRNA molecules per cell (a reasonable order-of-magnitude estimate, given that non-human primate ileum cells had a maximum of 10 ACE2 unique molecules per cell observed via sequencing and an average of 1.93 molecules per cell in expressing cells, see Figures 3B and 3C), our experimental platforms have a minimum likelihood of 41% to capture and reverse transcribe at least one ACE2 mRNA molecule in any given individual cell.
T852 46648-46889 Sentence denotes This likelihood rapidly increases if we estimate higher efficiencies for improved scRNA-seq technologies (e.g., 67% likelihood within any individual cell at 20% capture/transcription efficiency, 76% likelihood at 25% efficiency, Figure S6A).
T853 46890-47197 Sentence denotes Thus, while transcript drop-out may reduce the fraction of positive cells, with the capture and transcription efficiencies of improved single-cell technologies, the impact is likely to be minor (reads are likely underestimated by up to a factor of ∼2.5x), given a sufficient depth of sequencing (see below).
T854 47198-47385 Sentence denotes We note that this impacts both clusters deemed to contain and not contain ACE2+ cells, and suggests our percentages are likely lower bounds for true expression (within a factor of ∼2.5x).
T855 47386-47565 Sentence denotes Next, we examined the probability of sequencing an ACE2 transcript as a function of read depth and ACE2’s fractional abundance in each single cell within our sequencing libraries.
T856 47566-48106 Sentence denotes First, across two different tissues (non-human primate ileum and lung, representing a high expresser of ACE2 and low expresser, respectively), we calculated the proportion of unique ACE2 molecules in our ACE2+ cells (defined as any cell with at least 1 UMI aligning to ACE2) as a fraction of total reads within individual cells to provide an order-of-magnitude estimate for average ACE2 abundance in our single-cell sequencing libraries (i.e., the probability that a read within a cell corresponds to a unique molecule of ACE2, Figure S6B).
T857 48107-48448 Sentence denotes We highlight that by calculating probabilities based on ACE2 unique molecules divided by an individual cell’s total reads, we are providing a conservative estimate for the probability of observing ACE2 as a function of sequencing depth (e.g., as compared to basing these probabilities on ACE2 non-UMI-collapsed reads divided by total reads).
T858 48449-48591 Sentence denotes Next, we obtained information on the number of reads in these cell populations to provide estimates of average sequencing depths (Figure S6C).
T859 48592-48876 Sentence denotes Using the mean fractional abundances of ACE2 from each tissue (Figure S6B) and the mean read depths for all genes (Figure S6C), we calculated the probability of detecting at least 1 ACE2 molecule (i.e., P(detecting > 0 ACE2 molecules) = 1 - (1 - ACE2 fractional abundance)Read depth).
T860 48877-49109 Sentence denotes This results in a 93.7% probability in ileum-derived cell libraries that contain ACE2, and a 76.0% probability for lung-derived cell libraries, indicating that our sequencing depths are sufficient to detect ACE2+ cells (Figure S6D).
T861 49110-49339 Sentence denotes To further evaluate whether our ability to detect ACE2+ cells was an artifact of sequencing depth, we compared the number of ACE2+ cells in a cluster to the mean number of reads across all cells in that same cluster (Figure S6E).
T862 49340-49673 Sentence denotes We did not observe any significant correlation: the ileum cell cluster with the highest number of ACE2+ cells had the lowest sequencing depth of all ileum clusters, and the lung cell cluster with the highest number of ACE2+ cells was approximately average in its read depth (on a log-log scale, Pearson’s r = −0.31, non-significant).
T863 49674-50205 Sentence denotes Further, when comparing ACE2+ cells to ACE2- cells within a given tissue, we did not observe a positive correlation between read depth and ACE2 status (i.e., mean ± standard error of the mean, SEM, reads among all lung cells = 28,512 ± 344; mean ± SEM reads among ACE2+ lung cells = 28,553 ± 2,988; mean ± SEM reads among all ileum cells = 14,864 ± 288; mean ± SEM reads among ACE2+ ileum cells = 10,591 ± 441, full statistics on cell depth among ACE2+ cells compared to ACE2- cells of the same cell type can be found in Table S9).
T864 50206-50349 Sentence denotes Thus, we can be confident that the observed differences in ACE2+ proportions across clusters are not driven by differences in sequencing depth.
T865 50350-50472 Sentence denotes Finally, we investigated how observed differences in ACE2+ proportions across clusters might be affected by cell sampling.
T866 50473-50895 Sentence denotes Using the proportion of ACE2+ cells in a “typical” cluster annotated as being ACE2 positive (i.e., 6.8% in non-human primate type II pneumocytes, Figure 1), we calculated the cluster sizes needed to be confident that the probability of observing zero to a few positive cells is unlikely to have arisen by random chance (probabilities calculated under a negative binomial distribution with parameter p = 0.068, Figure S6E).
T867 50896-51081 Sentence denotes We found that as cluster sizes approach and exceed 100 cells, the probability of observing zero to a few positive cells rapidly approaches zero, if we assume 6.8% of cells are positive.
T868 51082-51699 Sentence denotes Further, to examine our confidence in estimating an approximate upper bound (ignoring the impact of protocol inefficiencies discussed above) for the fraction of cells positive in a cluster as a function of the number of cells in that cluster, we also calculated the probability of observing zero (and its complement, probability of observing at least 1) ACE2+ cells as a function of cluster size across true positive proportions ranging from 0.1% to 10% (probabilities calculated under a negative binomial distribution with parameter p = 0.001 to 0.1, representing hypothetical proportions of ACE2+ cells Figure S6F).
T869 51700-51940 Sentence denotes Given our typical cluster sizes (on the order of hundreds of cells, exact values provided in Table S9), we find that for us to observe 0 ACE2+ cells in a cluster due to sampling artifacts, the fraction of true positives must be ∼1% or less.
T870 51941-52137 Sentence denotes Thus, these complementary approaches demonstrate that our observed variations in ACE2+ cell proportions across clusters likely reflect underlying biological differences, rather than random chance.
T871 52139-52158 Sentence denotes Statistical Testing
T872 52159-52380 Sentence denotes Parameters such as sample size, number of replicates, number of independent experiments, measures of center, dispersion, and precision (mean ± SEM) and statistical significances are reported in Figures and Figure Legends.
T873 52381-52433 Sentence denotes A p value less than 0.05 was considered significant.
T874 52434-52598 Sentence denotes Where appropriate, a Bonferroni or FDR correction was used to account for multiple tests, alternative correction methods are noted in the figure legends or Methods.
T875 52599-52744 Sentence denotes All statistical tests corresponding to differential gene expression are described above and completed using R language for Statistical Computing.