title: GSM8008844: C3, replicate 1, scRNAseq; Rattus norvegicus; RNA-Seq
library_construction_protocol: Fresh rat lung tissues were obtained from rats exposed or unexposed to gas explosions. The tissues were immediately transferred into sterile, RNase-free culture dishes containing calcium- and magnesium-free PBS (CMF-PBS) on ice. They were then dissected into ~0.5 mm2 pieces, rinsed in CMF-PBS to remove blood and fat, and dissociated into single-cell suspensions using 0.35% collagenase IV, 5.2 mg/ml papain, and 120 U/ml DNase I in a 37°C shaking water bath (100 rpm, 20 min). Digestion was stopped with CMF-PBS + 10% FBS, followed by 5-10 pipetting passes through a Pasteur pipette. The cell suspension was filtered through stacked 70-30 μm pore cell strainers and centrifuged (300g, 5 min, 4°C). The pellet was resuspended in PBS + 0.04% BSA and treated with red blood cell lysis buffer (130-094-183, 10X) for 2-10 min at room temperature or on ice. After washing via centrifugation (300g, 5 min, room temperature), dead cells were removed using Dead Cell Removal MicroBeads (130-090-101). The cells were washed twice more with PBS + 0.04% BSA via centrifugation (300g, 3 min, 4°C) before final resuspension in the same buffer. Cell viability exceeding 85% was confirmed by trypan blue exclusion. Cells were counted on a hemocytometer or with a Countess II Automated Cell Counter and resuspended to 700-1,200 cells/μl. Single-cell suspensions were loaded onto the 10x Chromium Controller to capture no less than 5,000 single cells per the manufacturer's instructions (10X Genomics Chromium Single-Cell 3' kit v3). Subsequent cDNA amplification and library construction steps followed the standard protocol. Libraries were sequenced by LC-Bio Technology Co. Ltd. (Hangzhou, China) on an Illumina NovaSeq 6000 system (150 bp paired-end multiplexed run) to a minimum depth of 20,000 reads per cell.