title: GSM8114856: Spool, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Wild type C57Bl/6 mice (10 weeks old) were treated systemically for seven days with bleomycin (100 U/kg) using subcutaneously implanted osmotic minipumps (ALZET 1007D; DURECT Corporation, Cupertino, CA; containing 100 µl, designed to deliver contents at 0.5 µl/h for 7 days). Control mice were treated with saline vehicle. Pumps were removed on day 7 per manufacturer's instructions. On day 21, mice were sacrificed by isoflurane overdose and the lungs harvested. The rib cage was opened to expose the lungs. Mice were systemically perfused via the left ventricle with PBS. The left lobe was then tied and cut off. Lung tissue was washed in cold DMEM and minced into small pieces using fine surgical scissors. The tissue from each mouse was resuspended in 5 ml of collagenase (1 mg/ml, Roche #10103578001) and shaken at low speed for 45 min at 37°C. Digestion was stopped by addition of 10 ml of DMEM/10% FBS. The sample was passed through a 40 μm cell strainer to prepare a monodisperse cell suspension. Cells were collected by centrifugation (1200 rpm, 10 min, 4°C, Beckman GS-6R centrifuge) and the cell pellets resuspended in 1 ml ACK lysing buffer (LONZA #10-548E) for 5 min to remove red blood cells. Lung cells were then collected by centrifugation, washed with PBS, and collected again. Single-cell suspensions from six control saline-treated mice and six bleomycin-treated mice were each pooled in equal numbers prior to loading onto a 10X Genomics Next GEM Chip B and emulsified with Single Cell 3' GEM beads using a Chromium™ Controller (10x Genomics). Library was performed according to the manufacter's instruction.(single cell 3' protocol, 10x Genomics). Cells collected were counted; 50,000 cells from each sample were labeled in 200uL of PDB +1% BSA. Mixed well by repeated gentle pipetting. Incubated on ice for 30mins. Labeled cells washed 2X w/1.5ml PBS +1% BSA(pelleted between washes at 500 tcf x 5 min @RT). After final wash,labeled cells resuspended in ~50uL PBS + 1%BSA and counted. Processing on the 10x Genomics Single-Cell Protocol by the MUSC Translation Science Lab. Libraries were constructed using the Chromium Single Cell 3' Library Construction Kit (10x Genomics, v3.1) and sequenced at Vanderbilt's Next Gen Sequencing Core (Illumina NovaSeq 6000).