Transcriptome analysis of three cell lines: EBV-transformed B-cell line e9453, HEK293FT, and CaCo2

Total RNA was prepared from each 6 × 10⁶ cells using 1.5 ml TRIzol. RNA was used for library generation after assessment for purity by UV-VIS spectrometry (NanoDrop) and for integrity by Bioanalyzer 2100 (Agilent Technologies). RNA-seq libraries were prepared from 100 ng of total quality-controlled RNA with a strand-specific protocol (RNA-seq complete kit, NuGEN). In brief, RNA was reverse transcribed with a reduced set of hexamer primers, avoiding excessive representation of rRNA in the cDNA. Second strand cDNA synthesiswas performed in the presence of dUTP. After ultrasonic cDNA fragmentation, end repair Illumina-compatible adapters were ligated. Adapters contained uracil in one strand, allowing complete digestion of the second strand–derived DNA. After strand selection, the libraries were amplified, assessed for correct insert size on the Agilent Technologies Bioanalyzer, and diluted to 10 nM. Barcoded libraries were mixed in equimolar amounts and sequenced on a total of three lanes on a Genome Analyser IIx (Illumina) in single-read mode with a read length of 84 bp. Raw reads were demultiplexed by sorting for and simultaneously trimming a leading 4-bp barcode sequence at the 5′-end of each read. Obtained 80-bp reads were mapped to the hg19 release of the human genome using spliced-read mapper Tophat and the UCSC gene annotation. Mapped reads overlapping with known genes were counted with HTseq count, available from the developers of DESeq.