title: ScRNA-seq analysis of cultured primary CAFs (n=3) and NAFs (n=1)
library_construction_protocol: 3 human CAF lines from human CRC and one 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 three passages prior to scRNA-sequencing. 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. Cultivated fibroblasts were trypsinized, counted and loaded onto a 10x Chromium Next GEM Chip following the Chromium Next GEM Single Cell 3´user guide (10x Genomics). Amplified cDNA was used for 3' RNA-seq library generation, RNA-seq libraries were prepared following the manufacturer's user guide (10x Genomics).