GSM8003558: ClubTracer, bleo injuryed, scRNAseq; Mus musculus; RNA-Seq
After the mice were killed, the lungs were perfused using cold PBS through the right ventricle to wash out the blood cell and then the protease solution (collagenase type I (500U ml−1; Gibco 17100–017), elastase(4U ml−1; Worthington Biochemical Corporation LS002279), dispase (5 U ml−1; BD Biosciences 354235) and DNase I (0.33 U ml−1; Worthington Biochemical Corporation LS002139) in DMEM (Gibco, 11965092)) was injected into the lungs through the trachea to the lung. After that, the lungs were minced into small pieces and incubated in 4 ml protease solution for 30 min at a 37°C shaker with frequent agitation. Subsequently, the tissue pieces were dissociated by pipetting and centrifuged at 1000 x g for 5 min at 4°C. After washing with DMEM, the pellet was resuspended in 3 ml 0.1% Trypsin-EDTA (Gibco, 25200072) with DNase I and incubated for 20 minutes at a 37 °C shaker with agitation and pipetting. Then the DMEM with 10% FBS was added to terminate the digestion and the cells were filtered sequentially through 100 μm strainers and centrifuged at 1000 x g for 5 min at 4°C. The cells isolated from bleomycin-treated mice were sorted using Fluorescence-Activated Cell Sorting (FACS) based on their cell lineage, relying specifically on fluorescent signals (AT2-Tracer: ZsGreen+; BASCs-Tracer: ZsGreen+, tdTomato+; Club-Tracer: tdTomato+). Cells with distinct lineages were loaded into the 10x Chromium controller to generate GEMs respectively, which were subsequently processed into single-cell 5ʹ gene expression libraries using the Chromium Single Cell 5ʹ Library Kit (v2 Chemistry). All samples were pooled for sequencing on the Illumina NovaSeq 6000 PE150 platform.