GSM4730264: Fine needle aspiration - P5; Homo sapiens; RNA-Seq
Biopsies were minced with sterile surgical scalpels to 0.5-1 mm fragments in approximately 1 ml of media. After centrifugation for 5 minutes at 125 g, cells were resuspended in DMEM with 0.5 mg/ml Liberase TH (Sigma-Aldrich) and 1% penicillin-streptomycin (Corning) and incubated in an orbital shaker for 15 min at 37°C. Liberase was quenched with equal volume of 1% BSA in DMEM followed by centrifugation for 5 minutes at 125 g. Cells were resuspended in 2 ml Accutase (Sigma-Aldrich) and incubated in an orbital shaker for 15 minutes at 37°C. Organoids were dissociated by incubating with TrypLE Express (Thermo Fisher) for 5-15 minutes at 37°C followed by manual disruption. Dissociated cells were passed through a 40 μm strainer and centrifuged for 5 minutes at 125 g. Isolated cells were resuspended in 0.04% BSA in phosphate buffered saline (Corning) for subsequent viability analysis and counting. Single-cell transcriptomic amplification and library preparation was performed using the 5' or 3' Library Construction Kit (10x Genomics) following the manufacturer's recommendations. Amplification and library prep of the PM sample was performed using the SureCell WTA 3' Library Prep Kit for the ddSEQ System following the manufacturer's recommendations (Illumina, Cat# 20014279). single cell RNA sequencing