GSM9085032: cMYC_lucOSOE_Tp53KO_EV; Mus musculus; RNA-Seq
Tumors were cut into small pieces in ice-cold perfusion buffer and transferred to gentleMACS C tubes with four rounds of dissociation in digestion buffer with 0.5 mg/ml Collagenase IV and 0.1 mg/ml DNaseI using gentleMACS dissociator according to the manufacturer's instructions. The cell suspensions were passed through 70 µm cell strainers prior to loading to the Percoll gradient mix to create a gradient. After centrifugation, the pellets were subjected to red blood cell lysis and resuspended in 2% FBS-PBS FACS buffer. Isolated cells were stained with an anti-CD45-APC antibody  for 45 minutes on ice. LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit was used to exclude dead cells. Isolated cells were analyzed. CD45+ cells were sorted on a BD FASCAriaIII Flow Cytometer. The cells were gated on viability (without doublets via SSC-A SSC-H gating and dead cells) for subsequent scRNA-sequencing. The sorted live CD45+ cells stored in ice-cold FACS buffer were immediately processed according to manufacturer's instructions by encapsulation into single cell gel beads (GEMs) and unique molecular identifier (UMI) barcoding using the Chromium Next GEM 3' v3 chemistry Single Cell kit (10× Genomics) at The Beijing Genomics Institute. Libraries were prepared and sequenced on a NovaSeq platform (Illumina)
GEO
GSM9085032_r1