GSM2453556: Col_E_RNAseq1_PC; Homo sapiens; RNA-Seq
Total RNA was isolated using the RNeasy Kit (Qiagen) and included lysing in 350 μL RLT lysis buffer and the optional DNase treatment. cDNA libraries were prepared from 50 nanograms of total RNA and indexed with Illumina’s TruSeq RNA Sample Preparation Kit v2 (RS-122-2001 and RS-122-2002).  Final indexed cDNA libraries were pooled with 12 uniquely indexed TruSeq cDNA libraries for a total of 6 samples per lane.  Multiplexed samples were sequenced on an Illumina HiSeq 2500 with a single 51 bp read and a 7 bp index read. Base-calling and demultiplexing were performed using Casava (v1.8.2). Sequences were filtered and trimmed to remove low quality reads, adapters, and other sequencing artifacts. The remaining reads were aligned to 19084 Refseq genes extracted from the Illumina iGenomes reference, selecting only those with ‘NM_’ annotations.  Bowtie (v 0.12.9) was used for alignment, allowing two mismatches in a 28 bp seed [64]. Reads with more than 200 alignments were excluded from further analysis. RSEM (v1.2.3) was used to estimate isoform and relative gene expression levels (transcripts per million or “TPM”)