GSM8634609: Pooled mesenchymal cell samples [CMO_SC_S4]; Mus musculus; RNA-Seq
Mouse lungs were harvested following perfusion with 5 mL of PBS through the right ventricle. The tissue was mechanically dissociated using scissors and digested in an enzyme solution containing 0.25% Collagenase A, 1 U/mL Dispase II, and 2,000 U/mL DNase I in Hanks' Balanced Salt Solution. The tissue suspension was incubated at 37°C for 60 min with gentle trituration using a micropipette every 20 min. Following digestion, the cell suspension was filtered through a 100-μm cell strainer, washed with PBS, and resuspended in PBS containing 0.5% BSA. For whole cell samples, erythrocytes were removed by magnetic negative selection using rat anti-mouse TER-119 antibody followed by sheep anti-rat Dynabeads. For mesenchymal cell samples, cells were enriched by magnetic negative selection using rat anti-mouse antibodies against CD31, CD45, EpCAM, and TER-19, followed by sheep anti-rat Dynabeads. Following negative selection, both whole cell and mesenchymal cell samples from two biological replicates per genotype were multiplexed using the 10x Genomics 3' CellPlex Kit Set A. Sample tags were assigned as follows: whole cell samples (WT_1: CMO301, WT_2: CMO302, Tubb3-/-_1: CMO303, Tubb3-/-_2: CMO304) and mesenchymal cell samples (WT_1: CMO305, WT_2: CMO306, Tubb3-/-_1: CMO307, Tubb3-/-_2: CMO308). The labeled samples were then pooled according to sample type (whole cell or mesenchymal cell) and stained with DAPI (0.1 μg/mL) prior to cell sorting. For the pooled whole cell sample, live cells were sorted. For the pooled mesenchymal cell sample, MCAM+ PDGFRα- (mural cells) and MCAM- (non-mural cells) populations were sorted and mixed at a 1:1 ratio to achieve mural cell enrichment. Single-cell isolation, RNA capture, cDNA preparation, and library preparation were performed according to the manufacturer's protocol (Chromium Next GEM Single Cell 3' Reagent Kit, v3.1 chemistry, 10x Genomics).