title: GSM5467104: SPECTRUM-OV-007_S1_CD45P_BOWEL; Homo sapiens; RNA-Seq
library_construction_protocol: Dissociation: Tumor tissue was immediately processed for tissue dissociation. Fresh tissue was cut into 1 mm pieces and dissociated at 37°C using the Human Tumor Dissociation Kit (Miltenyi Biotec) on a gentleMACS Octo Dissociator. After dissociation, single cell suspensions were filtered and washed with ACK (Ammonium-Chloride-Potassium) Lysing Buffer. Cells were stained with Trypan Blue and cell counts and viability were assessed using the Countess II Automated Cell Counter (ThermoFisher) (for detailed protocol see 40). Sorting: Freshly dissociated cells were stained with a mixture of GhostRed780 live/dead marker (TonBo Biosciences) and Human TruStain FcX™ Fc Receptor Blocking Solution (BioLegend). The stained samples were then incubated and stained with Alexa Fluor® 700 anti-human CD45 Antibody (BioLegend). Post staining, they were washed and resuspended in RPMI + 2% FCS and submitted for cell sorting. The cells were sorted into CD45 positive and negative fractions by fluorescence assisted cell sorting (FACS) on a BD FACSAria™ III flow cytometer (BD Biosciences). Positive and negative controls were prepared and used to set up compensations on the flow cytometer. Cells were sorted into tubes containing RPMI + 2% FCS for sequencing. Flow sorted tumor cells were stained with Trypan blue and Countess II Automated Cell Counter (ThermoFisher) was used to assess both cell number and viability. Following QC, the single cell suspension was loaded onto Chromium Chip B (10x Genomics PN 2000060). GEM generation, cDNA synthesis, cDNA amplification, and library preparation of 1,400-5,000 cells proceeded using the Chromium Single Cell 3' Reagent Kit v3 (10x Genomics PN 1000075) according to the manufacturer's protocol. cDNA amplification included 12 cycles and 0.4-419ng of the material was used to prepare sequencing libraries with 8-14 cycles of PCR.