title: GSM1471693: shScr_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using RNeasy Mini Kit according to the manufacturer's protocol. Total RNA was quantified using the Quant-iT RiboGreen RNA Assay Kit and normalized. Libraries were prepared with an automated variant of the Illumina TruSeq mRNA Sample Preparation Kit that preserves strand orientation of the RNA transcript. This method uses oligo-dT beads to select mRNA from the total RNA sample, followed by heat fragmentation and cDNA synthesis from the RNA template. The resulting cDNA then goes through library preparation (end repair, base 'A' addition, adapter ligation, and enrichment) using indexed adapters for multiplexing. After enrichment, the libraries were quantified by qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then equimolar pooled. Pooled libraries were normalized and denatured using 0.2 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer's protocols on an Illumina HiSeq 2500 at the Broad Institute Specialized Service Facility. Each run generated 76bp paired-end reads with 8bp barcodes. Two biological replicates were sequenced for shScr- and shIR-CHB8 hESCs.