GSM5076108: polarized human airway epithelial cultures (HAEC); Homo sapiens; RNA-Seq
6-week-old human airway cultures were used for the single-cell RNA-seq analysis.  The apical surface was washed once to remove mucus by adding 100 µL of PBS and incubating for 30 min at 37°C. Cells were dissociated by cutting out the transwell membrane and incubating it in 700 µL TrpLE 10x (Thermo Fisher cat no.  A1217701) for 30 min rocking at 37°C. To increase dissociation cells were pipetted through wide-bore tips every 10 min during the incubation time. When cells were visually dissociated 700µl of ALI maintenance medium supplemented with 0.1% Pluronic (Thermo Fisher cat. No. 24040032) was added and cells were carefully pipetted again using wide-bore tips. The cell suspension was centrifuged through a 10 µM filter at 300 x g for 5 min to break up any remaining cell clumps. The cell pellet was washed once with 200 µL ALI maintenance medium supplemented with 0.1% Pluronic before cell number and viability was assed using the Countess II (Thermo Fisher) to calculate cell numbers used in the following steps for single-cell RNA-seq analysis. Single cell transcriptome profiling of dissociated organoids was carried out using the Chromium Next GEM Single Cell 5' Library & Gel Bead Kit and Chromium controller (10X genomics). To enable multiplexing and doublet detection, cells were stained with barcoded antibodies described previously (Mimitou et al., 2019). Briefly, approximately 200,000 cells per sample were resuspended in staining buffer (PBS, 2% BSA, 0.01% Tween) and incubated for 10 minutes with Fc block (TruStain FcX, Biolegend; FcR blocking reagent, Miltenyi). Cells were then incubated with barcoded hashing antibodies for 30 min at 4 °C. After staining, cells were washed 3 times in staining buffer. After the final wash, cells were resuspended in PBS + 0.04% BSA, filtered, and counted. Cells were pooled and loaded onto the Chromium chips. For each lane, we pooled 5 samples, ~10,000 cells per sample (we might also want to just include a table that shows how the samples were pooled, how many cells we were targeting, etc). The single-cell capturing, barcoding, and cDNA library preparation were performed using the Chromium Next GEM Single Cell 5' Library & Gel Bead Kit by following the protocols recommended by the manufacturer. HTO additive oligonucleotide was spiked into the cDNA amplification PCR and the HTO library was prepared as described previously.   The Cellranger software suite (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) from 10X was used to demultiplex cellular barcodes, align reads to the human genome (GRCh38 ensemble, http://useast.ensembl.org/Homo_sapiens/Info/Index) and perform UMI counting. From filtered counts HTODemux was used to demultiplex hash-tagged samples and Seurat1 version 3.1.3 was used to process the single cell data including dimension reduction, UMAP representation and differential expression to identify cell type specific markers determined by wilcox test.