design: Bulk RNA-seq analysis of 6 CAFs and 3 NAFs from human colorectal carcinoma
library_construction_protocol: 6 human CAF lines from human CRC and 3 matching NAFs from uninvolved healthy colon were isolated as described in Naschberger et al, JoVE2018 and Schellerer et al, Oncol Rep 2014 and propagated in culture for up to two passages prior to RNA extraction. Fibroblasts were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS, Sigma-Aldrich, #S0615) 1% L-Glutamin (Thermo Fisher, #25030-024) and 1% Penicillin/Streptomycin (Gibco, #15140-122) at 37°C, with 8.5% CO2 and 95% humidity. RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) following the manufacturers instruction. For each sample, a cDNA library was constructed using the TruSeq Stranded mRNA Kit (Illumina).