RNA-Seq of HT-29 colon adenocarcinoma cells treated with 5-FU, 72h, 20 microM
Total RNA samples were isolated from HT-29 cells untreated or treated with 20 µM 5-FU for 72 h, using the MagNA Pure Compact Instrument (Roche, Switzerland) according to the manufacturer's instructions (the procedure included DNase treatment). Purified RNA samples were quantified with Qubit 2.0 Fluorometer (Thermo Fisher Scientific, USA) and samples quality was estimated by the calculation of RNA Integrity Number (RIN), using Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA samples with the RIN higher than 8.0 were used for subsequent analysis. Total RNA (1 μg) from each sample was subjected to enrichment of polyadenylated RNA  and separation from rRNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, USA). сDNA library preparation was carried out using NEBNext Ultra Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (New England Biolabs, USA) according to manufacturer's instructions. The quality and concentration of cDNA libraries was assessed as described above, and cluster densities were optimized by qPCR, using Rotor-Gene Q 5 plex platform (Qiagen, Germany). Obtained cDNA libraries were sequenced in triplicate on the Illumina NextSeq 500 platform under the 2 × 43 bp paired-end model, yielding 170M mapped reads per experiment.