RNA-seq TIF-IA siRNA DNA Rep 2
Total RNA was isolated and DNAse I treated on RNeasy columns (74106; Qiagen). Illumina Truseq stranded poly(A)-selected mRNA libraries were prepared, multiplexed and sequenced in single-read mode (1 x 50 bp) on an Illumina HiSeq 2500 by the New York University Genome Technology Center. Per-sample FASTQ files were generated using the bcl2fastq2 Conversion software (v2.20) to convert per-cycle BCL base call files outputted by the sequencing instrument into the FASTQ format. Between 30 million and 56 million single-end reads were obtained per sample. The alignment program, STAR (v2.4.5a), was used for mapping reads of 12 human samples to the human reference genome hg19 and the application FastQ Screen (v0.5.2) was utilized to check for contaminants. The software, featureCounts (Subread package v1.4.6-p3), was used to generate the matrix of read counts for annotated genomic features. For the differential gene statistical comparisons between groups of samples contrasted by TIF-IA, UBF, Control, dsDNA, and non-dsDNA conditions, the DESeq2 package (Bioconductor v3.3.0) in the R statistical programming environment was utilized.