GSM5226826: ctl_o2; Homo sapiens; RNA-Seq
Lung tissue was cut into pieces and incubated with 2 mg/ml collagenase I, 2 mg/ml collagenase IV and 0.2 mg/ml DNase I (Roche) in RPMI 1640 for 40 min at 37℃ with 150 rpm shaking. The resulting cell suspension was filtered through a 70-μm sterile nylon strainer and centrifuged at 400 g for 5 min at 4℃. The cell pellet was resuspended in 1 ml RBC lysis buffer (0.15 mol/l NH4Cl, 10 mmol/l KHCO3, 0.1 mmol/l EDTA) and lysed for 10 min at room temperature. Cells were centrifuged at 400 g for 5 min at 4℃. The cell pellet was resuspended in 400 μl PBS for cell sorting by Countstar (IC1000). Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual microwells. Reverse transcription was performed on a ThermoMixer® C (Eppendorf) at 1,200 rpm and 37°C for 45 minutes. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200. scRNA-seq libraries were constructed using BD Rhapsody™ WTA Amplification Kit (BD, 633801). The libraries were sequenced on Illumina Novaseq6000 sequencer for at least 50,000 150 bp pair-end reads per cell.