title: Comparison of gene expression between 3 Renal Cell Carcinoma spheroids
design: Comparison of gene expression between 3 Renal Cell Carcinoma spheroids
library_construction_protocol: 786-O cell line (RRID: CVCL_1051) (ATCC CRL-1932)was maintained in RPMI-1640 medium, completed with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. RCC10 (RRID: CVCL_6265) cell line, provided by Gilles Pagès and Sandy Giuliano (University of Nice Sophia Antipolis), and RCC7 (RRID: CVCL_E057) cell line, provided by Joel Le Maoult (St-Louis hospital/CEA Paris-Saclay) were cultured in complete DMEM (Gibco).RCC cells were counted and coated with magnetic nanoparticles (NanoShuttle, Greiner) at 1 µL per 40,000 cells through three centrifugation/resuspension cycles (1000 rpm - 5 min). Cells were then seeded (3000 cells/well) in a 96-well U-bottom plate pre-coated with 20 mg/mL poly (2-hydroxyethyl methacrylate) (Sigma-Aldrich). The plate was incubated at 37°C, 5% CO2 for 3 days on a Spheroid-Drive support (Greiner) to form mature spheroid. RNA extracts from spheroids (786-O, RCC7 and RCC10) were obtained using the RNeasy Micro Kit (Qiagen) following manufacturer's instructions. Five spheroids were used for each extract. The RNA quantity and quality of the RNA was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and the concentration normalized to 100ng/ul. The generation of bulk RNA Barcoding libraries was performed using the MERCURIUSTM BRB-seq library preparation kit for Illumina and following the manufacturer's manual (Alithea Genomics, #10813).