Single-cell RNA-seq of LNCaP prostate cancer cells treated with Enzalutamide and Tazemetostat under hypoxic conditions
Single-cell RNA-seq of LNCaP prostate cancer cells treated with Enzalutamide and Tazemetostat under hypoxic conditions
LNCaP human prostate cancer cells were cultured under chronic hypoxia (2% O₂) in RPMI-1640 medium (Gibco #21875034) supplemented with 10% fetal bovine serum (ThermoFisher #26140079) and Penicillin/Streptomycin/Amphotericin B (ThermoFisher #15140122). Cells were harvested by trypsinization at experimental time points (Day 3, Day 6, Day 9), counted using a Luna-FL cell counter, and assessed for viability via trypan blue exclusion. Samples were maintained at 2% O₂ until processing. Single-cell suspensions were prepared following the 10x Genomics Chromium Next GEM Single Cell 3′ Kit v3.1 user guide. RNA molecules were captured within Gel Beads-in-Emulsion (GEMs) using barcoded oligo-dT primers. Reverse transcription within droplets generated cDNA tagged with unique molecular identifiers (UMIs) and cell barcodes. No separate bulk RNA extraction step was performed. cDNA synthesis and library preparation were performed using the 10x Genomics Chromium Next GEM Single Cell 3′ Kit v3.1 (PN-1000268/1000269) together with the 3′ Feature Barcode Kit (PN-1000262), Chromium Next GEM Chip G Single Cell Kit (PN-1000127/1000120), and Dual Index Kit TT Set A (PN-1000215). Libraries were constructed according to the manufacturer's instructions at the Babraham Institute Genomics Facility.