title: PXK cells (HT-29 single cell subline) with knocked down (experiment 2) LINC00973, replicate 2
design: Total RNA samples were isolated from HT-29 cells (PXK subline) untreated or treated with 20 M 5-FU for 72 h, using the MagNA Pure Compact Instrument (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturers instructions (the procedure included DNase treatment). Purified RNA samples were quantified with Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and samples quality was estimated by the calculation of RNA Integrity Number (RIN), using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). RNA samples with the RIN higher than 8.0 were used for subsequent analysis. Poly (A+) mRNA fraction was isolated from 1 g of total RNA samples using NEBNext poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, Massachusetts, USA). CDNA library preparation was carried out using NEBNext Ultra Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (New England Biolabs, Ipswich, Massachusetts, USA) according to manufacturers instructions. The quality and concentration of cDNA libraries were assessed as described above; cluster densities were optimized by qPCR, using Rotor-Gene Q 5 plex platform (Qiagen, Limburg, Netherlands). Obtained cDNA libraries were sequenced in triplicate on the Illumina NextSeq 500 platform under the 1x76 bp single-end model.