GSM5195681: Wild-Type lungs; Mus musculus; RNA-Seq
Lung cell dissociation. 25 mg fresh lung tissue was minced on ice and then shaken and triturated on ice for 9 minutes with 1.5 mL enzyme mix (90 uL of 100 mg/mL Collagenase A (Sigma), 150 L of 43 U/mL lyophilized Elastase (Worthington), 150 uL of 90 U/mL purified neutral Protease (Worthington), 7.5 uL of 1M CaCl2, 7.5 uL of 125 U/mL DNase I (Applichem), and 1095 uL of DPBS). After allowing to settle, the upper layer was filtered through a 30 uM filter and collected, followed by rinsing of filter with 0.04% BSA. Lich enzyme mix (1 uL of 0.5M EDTA, 100 L of 100 mg/mL Bacillus Licheniformis (Sigma), and 899 uL DPBS) was added to remaining tissue and triturated on ice for 10 minutes before being filtered and collected. Filter was again rinsed with 0.04% BSA, after which samples were lysed with RBC Lysis solution before being resuspended in 0.04% BSA. Following dissociation, cells were diluted to 1,000 cells/mL, and 15,000 cells from wild-type and MAS mice were each loaded in a separate lane of a Chromium Controller Instrument (10x Genomics). The 10x Genomics single cell 3' v3 assay protocol was followed as described until the cDNA amplification step.