GSM9630417: scRNA_GSC1ShPDLIM4; Mus musculus; RNA-Seq
The isolated human glioma stem cells (GSCs) were resuspended in digestion buffer (HBSS with Mg²⁺/Ca²⁺ supplemented with 2 mg/ml collagenase type Ⅰ and 40 U/ml DNase Ⅰ) and incubated at 37℃ for 30 minutes.The cells were centrifuged, washed, and resuspended in PBS, then stained with a Viability Dye. After another wash, the cells were resuspended in FACS buffer (HBSS without Mg²⁺/Ca²⁺, 2% v/v Fetal Calf Serum (FCS), 5 mM EDTA).Using normal human brain glial cells as the negative control, viable human glioma stem cells were sorted by FACS Jazz flow cytometer (Becton Dickinson) according to the presence or absence of mCherry signal.The sorted human glioma stem cells were washed with HBSS without Mg²⁺/Ca²⁺ and then resuspended in HBSS supplemented with 0.04% (w/v) Bovine Serum Albumin (BSA) for subsequent experiments. Single-cell suspension was added to the 10xChromium chip according to theinstructions for the 10xGenomics Chromium Single-Cell 3' kit (V3.1), with the expectation ofcapturing 8,000 cells. cDNA amplification and library construction were performedaccording to standard protocols. Libraries were sequenced by LC-Bio Technology (Hangzhou.hina) on an Illumina NovaSeg 6000 sequencing system (double-end sequencing, 150 bp) ata minimum depth of 20,000 reads per cell.