GSM9249185: Transbronchial lung biopsy 8 replicate 3; Homo sapiens; RNA-Seq
A cell suspension was prepared from transbronchial lung biopsies by digestion in 1.4ml DMEM containing 0.3 mg/μl liberase (Sigma), 70 μg/μl elastase (Worthington) and 3 U/ml dispase II (Sigma) at 37o C for 45 min, followed by the addition of 10μl DNAse (2.7 U/μl; Qiagen), 1μl 1M DTT, and 2μl 0.5M EDTA, and incubation for an additional 15 min. The cell suspension was strained through a 100-μm filter, centrifuged at 300xg for 10min, and resuspended in ~200μl residual buffer plus 10μl DNAse. Red blood cells were lyzed by adding 1ml ACK buffer (Gibco) and incubating on ice for 2 min. Following the addition of 10ml PBS with 1% BSA, centrifugation and resuspension in ~200μl residual buffer plus 10μl DNAse, the cells were blocked with FcR Blocking Reagent (Miltenyi) and stained 1:100 with CD326(EpCAM)-AF488 (Invitrogen), CD31-APC (Biolegend), CD45-APC (Biolegend), and CD235a-PE (Biolegend) for 1hr at 4 o C, strained through a 100-μm filter, and flow sorted on a BD Influx fluorescence-activated cell sorter (FACS). We positively sorted for complex, singlet cells and negatively sorted for DAPI and PE, which removed the dead cells and any remaining red blood cells. The FACS-sorted cells were collected as three populations: CD326/EpCAM positive (epithelial), CD31/CD45 positive (immune and endothelial), and those negative for either AF488 or APC fluorophore signals (enriched for fibroblasts). Using the cell count numbers obtained by FACS, each of the three populations of cells were mixed 1:1:1, using the population with the fewest number of cells as the limiting factor 10,000 cells were loaded on a 10X Genomics flow cell for encapsulation in individual droplets for lysis, reverse transcription, and barcoding of cDNA, followed by sequencing on an Illumina NovaSeq 6000. We performed additional quality control by visualizing transcript count, gene count, and percent mitochondrial genes for each cell cluster and by each sample.