LitCovid-sentences  

PMC:7252096 / 86975-102218 JSONTXT 11 Projects

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Id Subject Object Predicate Lexical cue
T617 0-14 Sentence denotes Method Details
T618 16-91 Sentence denotes Methods of Sample Collection and Tissue Preparation for Single-Cell RNA-Seq
T619 93-158 Sentence denotes NHP Ileum, Jejunum, Colon, Liver, Tonsil, Thymus, and Lung Tissue
T620 159-322 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 323-470 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 471-701 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 703-742 Sentence denotes NHP Lymphoid Organs, Bone Marrow, PBMCs
T624 743-915 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 916-1018 Sentence denotes LN-derived cells were resuspended in RPMI + 10% FBS, counted and prepared as a single cell suspension.
T626 1019-1139 Sentence denotes Spleen, bone marrow, and PBMCs were subjected to ACK lysis for 10 min at room temperature, quenched with RPMI + 10% FBS.
T627 1140-1299 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 1300-1412 Sentence denotes Cells were then resuspended in RPMI + 10% FBS, counted, and diluted for scRNA-seq using Seq-Well v1 (see below).
T629 1414-1458 Sentence denotes NHP Tuberculosis Infected Lung and Granuloma
T630 1459-1592 Sentence denotes Ten Mycobacterium tuberculosis infected (Martin et al., 2017) adult non-human primates (M. fascicularis) were included in this study.
T631 1593-1835 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 1836-1960 Sentence denotes Single cell suspensions were resuspended in RPMI + 10% FBS, counted and diluted for scRNA-seq using Seq-Well S3 (see below).
T633 1962-1979 Sentence denotes Human Lung Tissue
T634 1980-2132 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 2133-2402 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 2403-2559 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 2560-2768 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 2770-2781 Sentence denotes Human Ileum
T639 2782-2871 Sentence denotes Single-cell suspensions were collected from biopsies as described (Smillie et al., 2019).
T640 2872-3021 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 3022-3216 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 3217-3349 Sentence denotes Following digestion, cells were pelleted by centrifugation, subjected to ACK lysis for 3 min, and filtered through a 40 μm strainer.
T643 3350-3485 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 3487-3519 Sentence denotes Nasal Mucosa and Nasal Scrapings
T645 3520-3661 Sentence denotes Surgical samples from ethmoid sinus and nasal scraping of the inferior turbinate were processed as described (Ordovas-Montanes et al., 2018).
T646 3662-3892 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 3893-3950 Sentence denotes Samples were triturated and digestion quenched with EDTA.
T648 3951-4260 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 4262-4304 Sentence denotes Interferon Treatment of Mouse Nasal Mucosa
T650 4305-4438 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 4439-4538 Sentence denotes Respiratory and olfactory mucosa were isolated as in (Davidson et al., 2004, Dunston et al., 2013).
T652 4539-4681 Sentence denotes Briefly, using surgical tools under a dissecting microscope, the skull bones surrounding the nasal tissue of skinned mouse heads were removed.
T653 4682-4761 Sentence denotes The respiratory and olfactory mucosa were collected in RPMI media with 10% FBS.
T654 4762-4876 Sentence denotes Cells were digested in media containing Liberase TM (Roche) and DNase I (Roche) for 30 min at 37°C with agitation.
T655 4877-5017 Sentence denotes Cells were filtered using a 70 μm strainer, washed with EDTA-containing media to quench enzymatic digestion, and pelleted by centrifugation.
T656 5018-5194 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 5196-5221 Sentence denotes MHV68 Infected Mouse Lung
T658 5222-5306 Sentence denotes Mice were housed in individually ventilated cages during the MHV68 infection period.
T659 5307-5407 Sentence denotes MHV68 stocks were grown and quantified by plaque assay as previously described (Adler et al., 2000).
T660 5408-5533 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 5534-5619 Sentence denotes Prior to i.n. infection, mice were anesthetized with medetomidine–midazolam–fentanyl.
T662 5620-5756 Sentence denotes At the predetermined time points, mice were sacrificed by cervical dislocation and lung tissue was processed for subsequent experiments.
T663 5757-5984 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 5985-6081 Sentence denotes Single cells were harvested by straining the digested tissue suspension through a 70μm strainer.
T665 6082-6193 Sentence denotes After centrifugation at 300 x g for 5 min, single cells were counted, and prepared as a single cell suspension.
T666 6194-6325 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 6327-6366 Sentence denotes Nasal Washes during Influenza Infection
T668 6367-6881 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 6882-6996 Sentence denotes Samples were obtained by irrigation of each naris with up to 10 mL of saline, and collected in a single container.
T670 6997-7071 Sentence denotes The sample was then transported to the research laboratory for processing.
T671 7072-7212 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 7213-7312 Sentence denotes The material was strained using a 40 μm nylon cell strainer (Corning) into a 50 mL centrifuge tube.
T673 7313-7363 Sentence denotes Cells were pelleted at 1300 rpm for 10 min at 4°C.
T674 7364-7553 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 7554-7821 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 7822-7938 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 7940-7998 Sentence denotes Methods to Generate Single-Cell and Bulk RNA-seq Libraries
T678 8000-8011 Sentence denotes Seq-Well v1
T679 8012-8071 Sentence denotes Seq-Well was performed as described (Gierahn et al., 2017).
T680 8072-8190 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 8191-8297 Sentence denotes 15,000 cells were deposited onto the top of each PDMS array and let settle by gravity into distinct wells.
T682 8298-8393 Sentence denotes The array was gently washed with PBS, and sealed using a functionalized polycarbonate membrane.
T683 8394-8609 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 8610-9100 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 9101-9161 Sentence denotes Exonuclease digestion and PCR were carried out as described.
T686 9162-9296 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 9297-9460 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 9461-9669 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 9670-9798 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 9799-9992 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 9993-10010 Sentence denotes 20 bases, read 2:
T692 10011-10034 Sentence denotes 50 bases, read 1 index:
T693 10035-10043 Sentence denotes 8 bases.
T694 10045-10056 Sentence denotes Seq-Well S3
T695 10057-10144 Sentence denotes Seq-Well S3 modified the following protocol steps from v1, above (Hughes et al., 2019).
T696 10145-10235 Sentence denotes First, hybridization buffer was supplanted with 8% (v/v) polyethylene glycol (PEG, Sigma).
T697 10236-10360 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 10361-10613 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 10615-10624 Sentence denotes 10X v2 3′
T700 10625-10762 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 10763-11027 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 11028-11135 Sentence denotes An input of 6,000 single cells was added to each channel with a recovery rate of approximately 2,000 cells.
T703 11137-11145 Sentence denotes Drop-seq
T704 11146-11240 Sentence denotes Drop-seq experiments were performed according to the original protocol (Macosko et al., 2015).
T705 11241-11367 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 11368-11482 Sentence denotes Droplet emulsions were collected for 10-20 min/each prior to droplet breakage by perfluorooctanol (Sigma-Aldrich).
T707 11483-11603 Sentence denotes After breakage, beads were harvested and the hybridized mRNA transcripts reverse transcribed (Maxima RT, Thermo Fisher).
T708 11604-11680 Sentence denotes Exonuclease digestion and PCR were carried out as described (12 PCR cycles).
T709 11681-11847 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 11848-11938 Sentence denotes Single-cell libraries were sequenced in a 100 bp paired-end run on the Illumina HiSeq4000.
T711 11940-11967 Sentence denotes Smart-Seq2 for Bulk RNA-Seq
T712 11968-12070 Sentence denotes Population RNA-seq was performed as described (Ordovas-Montanes et al., 2018, Trombetta et al., 2014).
T713 12071-12367 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 12368-12700 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 12701-12795 Sentence denotes Libraries were purified using AMPure XP SPRI beads at a volume ratio of 0.8x followed by 0.9x.
T716 12796-12940 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 12941-13141 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 13142-13356 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 13357-13515 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 13517-13564 Sentence denotes Human and Mouse Basal Cell Cytokine Stimulation
T721 13565-13599 Sentence denotes Data represented in Figures 5A–5L:
T722 13600-13708 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 13709-13742 Sentence denotes Biolegend 574002), IL-17A (human:
T724 13743-13774 Sentence denotes Biolegend 570502), IFNγ (human:
T725 13775-13799 Sentence denotes Biolegend 570202; mouse:
T726 13800-13831 Sentence denotes Peprotech 315-05), IFNα (human:
T727 13832-13856 Sentence denotes Biolegend 592702; mouse:
T728 13857-13917 Sentence denotes Biolegend 752802), or IFNβ (mouse: R&D Systems 8234-MB-010).
T729 13918-13968 Sentence denotes Each condition was run as a biological triplicate.
T730 13969-14309 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 14310-14433 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 14434-14538 Sentence denotes Bulk RNA-seq was performed as described previously and summarized above (Ordovas-Montanes et al., 2018).
T733 14539-14742 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 14743-14825 Sentence denotes All samples met quality thresholds regarding genomic and transcriptomic alignment.
T735 14827-14854 Sentence denotes Western blot for human ACE2
T736 14855-14998 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 14999-15130 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 15131-15243 Sentence denotes Bands were normalized to GAPDH as loading control, and fold change was computed based on normalized ACE2 values.