GSM6732572: LenR_Len; Mus musculus; RNA-Seq
Tumors from mouse livers were harvested, cut into small pieces in ice-cold perfusion buffer (25mM HEPES pH7.4, 0.5mM EDTA in PBS) and transferred to gentleMACS C tube with four rounds of dissociation in digestion buffer with 0.5 mg/ml Collagenase IV and 0.1 mg/ml DNaseI (Roche) using gentleMACS dissociator. The cell suspensions were passed through 70 µm cell strainers prior to loading to percoll gradient mix to create the gradient. After centrifugation, the pellets were subjected to red blood cell lysis and resuspended in PBS supplemented with 2% FBS. Isolated cells were stained with anti-CD45-APC antibody for 45 minutes on ice. LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit was used for exclusion of dead cells. Isolated cells were analyzed and the live CD45+ subpopulation was sorted on a BD FACSAriaIII cell sorter The sorted live CD45+ cells stored in ice-cold FACS buffer were processed according to manufacturer's instructions by encapsulation into single cell gel beads and unique molecular identifier barcoding using the 10X Genomics Chromium Next GEM 3' v3 chemistry Single Cell kit at The Beijing Genomics Institute. Libraries were prepared and sequenced on Illumina NovaSeq platform.