GSM7951958: BLM2; Mus musculus; RNA-Seq
To obtain single cell solutions of tumor cells, pooled distal (Min, MSH2KO), distal and proximal (BLM) or proximal (BLM2) tumors were washed in HBSS, incubated in 0.25% trypsin-EDTA for 10 minutes at 37oC, followed by the addition of FBS to inactivate the trypsin. Next, the tumors were digested by incubation in liberase TM (0.05 mg/ml; Roche, #05401119001)+DNAse (0.2 mg/ml) in DMEM at 37oC for 2H while rotating. Following washing in HBSS and an additional incubation in 0.25% trypsin-EDTA for 10 minutes at 37oC, cells were resuspended in DMEM+10%FBS and filtered through a 40 mM mesh cell strainer. Cells were then washed with DPBS+0.1% BSA and viable cells were counted. Cells were resuspended at 1,000 cell per ml in DPBS+0.1% BSA All single cell preparations used for sequencing had a viability of > 80%. 10,000 cells per sample were targeted for input to the 10X Genomics Chromium system using the Chromium Next GEM Single Cell 30 Kit v3.1 at the Indiana University School of Medicine (IUSM) Center for Medical Genomics core