GSM8244384: Patient 15, PBMC CD45+, 5' GEX scRNAseq; Homo sapiens; RNA-Seq
Processing of fresh samples occurred within 30 min after surgical resection and transfer in ice-cold transport media. Tumor tissues were minced and single cell suspensions prepared by homogenizing the tissue in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) with 0.11 DMC U/mL neutral protease (NP, Nordmark Biochemicals, Germany) at 37°C for approximately 30 minutes in a shaker-incubator. Tissue dissociation was supported by gentle intermittent resuspension. Bone chips were continuously flushed with pre-warmed (37°C) 0.11 DMC U/mL NP in IMDM for 10–15 minutes in 15 mL canonical tubes to facilitate vital cell release. Cell suspensions were filtered by a 35 µm cell strainer. PBMC and BMMC were isolated from venous blood or from distal hip bone marrow. Aspirates were collected in 7.5 mL EDTA vacutainers or heparinized tubes, respectively. PBMC and BMMC were seprarated using the high-density medium Histopaque®-1077 by density centrifugation according to the manufacturer's suggestions.  For subsequent 10x Genomics processing, respective single cell suspensions were washed in PBS supplemented with 0.04% BSA and enriched for CD45+ cells by magnetic cell separation. Indicated samples underwent additional magnetic depletion of myeloid cells (CD14 negative flowthrough) prior to 10x Genomics processing. Samples were processed using the Chromium Next GEM Single Cell 3´ Reagent Kits v3.1 (Dual Index) (10x Genomics, #CG0000315 Rev C) or the Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) (#CG0000331 Rev E), following the manufacturer's instructions. Following a standard quality control (2100 Bioanalyzer, Agilent), paired-end sequencing of pooled libraries was performed using a NovaSeq 6000 system (Illumina).