GSM7963781
1x10^6 WT or Mga KO 4T1 cells were implanted into BALB/c mice and incubated for 3 weeks. Tumors were harvested, minced into small pieces on ice, and digested in DMEM supplemented with 5% FBS, collagenase IA (2 mg/ml, Sigma-Aldrich #C9891), hyaluronidase (1 mg/ml, Sigma-Aldrich #H3506), and dispase II (2 mg/ml, Sigma #D4693) for 45 mins with shaking at 37 °C. The tumor lysate was filtered with 70-mm strainer and washed twice with ice-cold PBS containing 2% FBS, then re-suspended in RBC lysis buffer (ThermoFisher) for 1 min at 37 °C to lyse red blood cells. After washing twice with ice-cold PBS containing 2% FBS, single-cell suspensions with high viability were obtained via sorting with DAPI staining Single cell Gene Expression Library was prepared according to Chromium Single Cell Gene Expression 3v3.1 kit (10x Genomics) by the Single Cell Genomics Core at Baylor College of Medicine. In Brief, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller (10x Genomics) to generate single cell GEMS (Gel Beads-In-Emulsions) where full length cDNA was synthesized and barcoded for each single cell. Subsequently the GEMS are broken and cDNA from each single cell are pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA is amplified by PCR. The amplified product is fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. Final library was generated by amplification.