title: GSM2720281: wtCSCs1 (A1_rep2); Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted using TRIzol Reagent. The extracted RNA integrity was measured with an Agilent 2100 Bioanalyzer, and a sample with an RNA Integrity Number greater than or equal to 8 was considered acceptable.   The libraries were generated using depleted RNA obtained from 1μg total RNA by TruSeq Sample Preparation RNA Kit according to the manufacturer’s without further modifications. The first step involves the removal of ribosomal RNA rRNA using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads that depletes samples of both cytoplasmic and mitochondrial rRNA. Following purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter  (Multiple indexing adapters were ligate to the ends of the ds cDNA). The products are purified and enriched with PCR to create the final cDNA library.   All libraries were sequenced on the Illumina HiSeq 1000 generating 100 bp paired end reads.