GSM7805184: L35146, LGMA; Homo sapiens; RNA-Seq
Minced tissue (1-3 mm) was digested with 3 -5 ml RMPI medium containing 0.05mg/ml TM Liberase (Sigma Aldrich, 5401119001) and 0.02 mg/ml DNAaseI (ThermoFisher, EN0521) at 37 ° C for 30 mins with rotation at 150 rpm. Cells were filtered through a 70 µM and 37 µM cell strainer to isolate single cells. The cell suspension was centrifuged at 500xg for 10 mins and resuspended with 5 ml ACK lysis buffer (K.D Medical, RGF3015) at room temperature for 3 mins to remove red blood cells. Microbeads (Miltenyi Biotec, 130-090-101) were used for dead cell removal. Cell viability and number were detected using a TC Automated Cell Counter (Bio-Rad Laboratories), and samples with at least 70% cell viability were processed Cells were captured on a Chromium controller (10xGenomics) using a single cell 3' Reagent kit V3.1 (10X Genomics, PN-1000121) targeting ~5000-10000 cell/sample