Single-Cell RNA-sequencing of lung mesenchymal cells at multiple time points after bleomycin injury

Mesenchymal cells (CD31-, CD45-, EpCAM-, Ter119-) were collected for 10x Genomics Chromium Single Cell v3.1 scRNA-seq from the lungs of Scube2-CreER/Rosa26-tdTomato double homozygous mice on day 0 , 7, 14, 21 after bleomycin treatment. Scube2-CreER, Rosa26-tdTomato double homozygous mice were treated with tamoxifen for two weeks. Bleomycin treatment was performed 2 weeks after the last tamoxifen treatment. Three biological replicates from day 0 (non-bleomycin-treated), 7, 14, and 21 samples were collected on the same day and tamoxifen/bleomycin treatment was scheduled accordingly to the collection day. After harvesting and dissociating left lungs, mesenchymal cells were enriched by magnetic negative selection with anti-CD31, CD45, EpCAM and Ter119-biotin antibodies (1:200) and Dynabeads MyOne Streptavidin T1 (40 ul / sample, Invitrogen). After magnetic negative selection, cells were stained with Streptavidin-APC/Cy7 (1:200) and DAPI (0.1 ug/ml). Approximately 2 x 10^5 lineage-APC/Cy7-negative cells were sorted for each sample. The sorted cells were counted and labeled with oligonucleotide tags for multiplexing using 10x Genomics 3’ CellPlex Kit. Tag assignment was as follows; day 0 (301, 302, 303), day 7 (304, 305, 306), day 14 (307, 308, 309), and day 21 (310, 311, 312). All 12 samples were pooled and 30,000 cells / lane were loaded onto 4 lanes of Chromium Next GEM Chip (10x Genomics). Chromium Single Cell 3’ v3.1 (10x Genomics) reagents were used for library preparation according to manufacturer’s protocol. The libraries were sequenced on Illumina NovaSeq 6000 S4 flow cell.