Method Details

Methods of Sample Collection and Tissue Preparation for Single-Cell RNA-Seq

NHP Ileum, Jejunum, Colon, Liver, Tonsil, Thymus, and Lung Tissue
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. 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. 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).

NHP Lymphoid Organs, Bone Marrow, PBMCs
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. LN-derived cells were resuspended in RPMI + 10% FBS, counted and prepared as a single cell suspension. Spleen, bone marrow, and PBMCs were subjected to ACK lysis for 10 min at room temperature, quenched with RPMI + 10% FBS. 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. Cells were then resuspended in RPMI + 10% FBS, counted, and diluted for scRNA-seq using Seq-Well v1 (see below).

NHP Tuberculosis Infected Lung and Granuloma
Ten Mycobacterium tuberculosis infected (Martin et al., 2017) adult non-human primates (M. fascicularis) were included in this study. 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). Single cell suspensions were resuspended in RPMI + 10% FBS, counted and diluted for scRNA-seq using Seq-Well S3 (see below).

Human Lung Tissue
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). 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). 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. 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).

Human Ileum
Single-cell suspensions were collected from biopsies as described (Smillie et al., 2019). 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. 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. Following digestion, cells were pelleted by centrifugation, subjected to ACK lysis for 3 min, and filtered through a 40 μm strainer. Following centrifugation, the cells were counted and prepared as a single cell suspension for scRNA-seq using 10X 3′ v2 (10X Genomics).

Nasal Mucosa and Nasal Scrapings
Surgical samples from ethmoid sinus and nasal scraping of the inferior turbinate were processed as described (Ordovas-Montanes et al., 2018). 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. Samples were triturated and digestion quenched with EDTA. 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).

Interferon Treatment of Mouse Nasal Mucosa
Mice received either 200ng of IFNα (Biolegend 752802) or saline intranasally (each group n = 2 mice), and were sacrificed 12 h later. Respiratory and olfactory mucosa were isolated as in (Davidson et al., 2004, Dunston et al., 2013). Briefly, using surgical tools under a dissecting microscope, the skull bones surrounding the nasal tissue of skinned mouse heads were removed. The respiratory and olfactory mucosa were collected in RPMI media with 10% FBS. Cells were digested in media containing Liberase TM (Roche) and DNase I (Roche) for 30 min at 37°C with agitation. Cells were filtered using a 70 μm strainer, washed with EDTA-containing media to quench enzymatic digestion, and pelleted by centrifugation. 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.

MHV68 Infected Mouse Lung
Mice were housed in individually ventilated cages during the MHV68 infection period. MHV68 stocks were grown and quantified by plaque assay as previously described (Adler et al., 2000). 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. Prior to i.n. infection, mice were anesthetized with medetomidine–midazolam–fentanyl. At the predetermined time points, mice were sacrificed by cervical dislocation and lung tissue was processed for subsequent experiments. 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). Single cells were harvested by straining the digested tissue suspension through a 70μm strainer. After centrifugation at 300 x g for 5 min, single cells were counted, and prepared as a single cell suspension. For Drop-seq, cells were aliquoted in PBS supplemented with 0.04% of bovine serum albumin at a final concentration of 100 cells/μl.

Nasal Washes during Influenza Infection
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). Samples were obtained by irrigation of each naris with up to 10 mL of saline, and collected in a single container. The sample was then transported to the research laboratory for processing. 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. The material was strained using a 40 μm nylon cell strainer (Corning) into a 50 mL centrifuge tube. Cells were pelleted at 1300 rpm for 10 min at 4°C. 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. 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. The final pellet was resuspended in up to 1 mL of media and quantified before performing scRNA-seq with Seq-Well v1.

Methods to Generate Single-Cell and Bulk RNA-seq Libraries

Seq-Well v1
Seq-Well was performed as described (Gierahn et al., 2017). Single cells were diluted to 15,000 cells in 200 μL RPMI + 10% FBS and deposited onto a pre-functionalized PDMS array. 15,000 cells were deposited onto the top of each PDMS array and let settle by gravity into distinct wells. The array was gently washed with PBS, and sealed using a functionalized polycarbonate membrane. 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. 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. Exonuclease digestion and PCR were carried out as described. Post-whole transcriptome amplification workup involved AMPure XP SPRI bead cleanup occurred at a 0.6 x volume ratio, followed by 0.8x. 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. 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. Amplified final libraries were purified twice with AMPure XP SPRI beads as before, with a volume ratio of 0.6x followed by 0.8x. 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: 20 bases, read 2: 50 bases, read 1 index: 8 bases.

Seq-Well S3
Seq-Well S3 modified the following protocol steps from v1, above (Hughes et al., 2019). First, hybridization buffer was supplanted with 8% (v/v) polyethylene glycol (PEG, Sigma). Second, after exonuclease digestion, bead-associated cDNA was denatured for 5 min in 0.2 mM NaOH with end over end rotation. 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.

10X v2 3′
Single cells were loaded onto 3′ library chips as per the manufacturers protocol for Chromium Single Cell 3′ Library (v2) (10X Genomics). 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. An input of 6,000 single cells was added to each channel with a recovery rate of approximately 2,000 cells.

Drop-seq
Drop-seq experiments were performed according to the original protocol (Macosko et al., 2015). Briefly, single cells (100/μl) were co-encapsulated in droplets with barcoded beads (120/μl, ChemGenes) at rates of 4000 μl/h. Droplet emulsions were collected for 10-20 min/each prior to droplet breakage by perfluorooctanol (Sigma-Aldrich). After breakage, beads were harvested and the hybridized mRNA transcripts reverse transcribed (Maxima RT, Thermo Fisher). Exonuclease digestion and PCR were carried out as described (12 PCR cycles). 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). Single-cell libraries were sequenced in a 100 bp paired-end run on the Illumina HiSeq4000.

Smart-Seq2 for Bulk RNA-Seq
Population RNA-seq was performed as described (Ordovas-Montanes et al., 2018, Trombetta et al., 2014). 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. 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. Libraries were purified using AMPure XP SPRI beads at a volume ratio of 0.8x followed by 0.9x. Library size was assessed using a High-Sensitivity DNA chip (Agilent Bioanalyzer), confirming the expected size distribution of ∼1,000-2,000 bp. 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. 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). 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.

Human and Mouse Basal Cell Cytokine Stimulation
Data represented in Figures 5A–5L: 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: Biolegend 574002), IL-17A (human: Biolegend 570502), IFNγ (human: Biolegend 570202; mouse: Peprotech 315-05), IFNα (human: Biolegend 592702; mouse: Biolegend 752802), or IFNβ (mouse: R&D Systems 8234-MB-010). Each condition was run as a biological triplicate. 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). All populations were lysed in 50 μL lysis buffer (RLT + 1% BME, QIAGEN and Sigma, respectively) and snap frozen on dry ice. Bulk RNA-seq was performed as described previously and summarized above (Ordovas-Montanes et al., 2018). 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%. All samples met quality thresholds regarding genomic and transcriptomic alignment.

Western blot for human ACE2
Established air-liquid interface cultures from bronchial brushings of four asthmatic patients were treated with 10ng/μL of human IFNγ for 24 h. Protein lysates were prepared, and anti-ACE2 human antibody (AF933 R&D goat polyclonal) was used to probe for ACE2 by western blot. Bands were normalized to GAPDH as loading control, and fold change was computed based on normalized ACE2 values.