GSM9267858: SPC-Cul3-KO-Bleomycin-D14-1; Mus musculus; RNA-Seq
Lung sample were digested in an enzymatic cocktail (collagenase I/dispase II) with DNase I using a gentleMACS Octo Dissociator. For single cell RNA sequencing lung tissue was filtered through 40 μm filters, and viable cells were counted using a Countess II automated cell counter (Thermo). Single cell barcoding and complementary DNA (cDNA) library preparation were performed according to the manufacturer's protocol (Single Cell 3′ Reagent Kits v3, 10x Genomics, USA), and run on the Chromium X. Reverse transcription was performed at 53 °C for 45 min and cDNA was amplified for 12 cycles using a BioRad C1000 Touch thermocycler. We performed cDNA size selection using SpriSelect beads (Beckman Coulter, USA) and cDNA quality was confirmed with an Agilent Bioanalyzer High Sensitivity DNA chip. DNA fragmentation, end-repair, A-tailing, and ligation of sequencing adapters were performed per the manufacturer's protocol (10x Genomics, USA). The cDNA libraries were sequenced on a NovaSeq 6000 platform.