GSM8745002: CD45 positive immune cells isolated from plasma of the rGBM patient; Homo sapiens; RNA-Seq
Single cell suspension was prepared from fresh surgically resected tumor tissue using the Adult Brain Dissociation Kit (Miltenyi Biotec) according to manufacturer's protocol. Tumor infiltrating leukocytes and peripheral immune cells were further isolated using biotinylated CD45 antibodies coupled with magnetic streptavidin microbeads (Miltenyi Biotec) form the tumor single cell suspension and PBLs, respectively. Cell number and viability were analyzed using Nexcelom Cellometer Auto2000 with AOPI fluorescent staining method. Sixteen thousand cells were loaded into the Chromium Controller (10X Genomics, PN-120223) on a Chromium Next GEM Chip G (10X Genomics, PN-1000120), and processed to generate single cell gel beads in the emulsion (GEM) according to the manufacturer's protocol. The cDNA and library were generated using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics, PN-1000286) and Dual Index Kit TT Set A (10X Genomics, PN-1000215) according to the manufacturer's manual. Quality control for constructed library was performed by Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and Qubit DNA HS assay kit for qualitative and quantitative analysis, respectively. The multiplexed libraries were pooled and sequenced on Illumina HiSeq 4000 sequencer with 2 × 50 paired-end kits using the following read length: 28 bp Read1 for cell barcode and UMI, and 90 bp Read2 for transcript.