title: Control HEK293 cells rep3
design: Strand specific, polyA-enriched RNA sequencing was performed as described earlier (Haack et al., 2013). Briefly, RNA was isolated from whole-cell lysates using the AllPrep RNA Kit (Qiagen) and RNA integrity number (RIN) was determined with the Agilent 2100 BioAnalyzer (RNA 6000 Nano Kit, Agilent). For library preparation, 1 g of RNA was poly(A) selected, fragmented, and reverse transcribed with the Elute, Prime, Fragment Mix (Illumina). A-tailing, adaptor ligation, and library enrichment were performed as described in the TruSeq Stranded mRNA Sample Prep Guide (Illumina). RNA libraries were assessed for quality and quantity with the Agilent 2100 BioAnalyzer and the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). RNA libraries were sequenced as 150 bp paired-end runs on an Illumina HiSeq4000 platform. The STAR aligner (Dobin et al., 2013)(v 2.4.2a) with modified parameter settings (--twopassMode=Basic) was used for split-read alignment against the human genome assembly hg19 (GRCh37) and UCSC knownGene annotation. To quantify the number of reads mapping to annotated genes we used HTseq-count (Anders et al., 2015) (v0.6.0). FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) values were calculated using custom scripts. Differential expression analysis was performed using the R Bioconductor package DESeq2 (Love et al., 2014).