title: GSM2769429: WT_HC_rep1; Mus musculus; RNA-Seq
library_construction_protocol: RNA-Seq libraries were generated from 300 ng of total RNA Illumina® TruSeq® RNA Sample Preparation Kit v2 (Part Number RS-122-2001). Briefly, following purification with poly-T oligo attached magnetic beads, the mRNA was fragmented using divalent cations at 94oC for 2 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Second strand cDNA synthesis followed, using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C) to create the cDNA library. Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman Coulter) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis. Sequencing was performed on the Illumina Genome Hiseq2500 as single-end 50 base reads following Illumina’s instructions.
attributes.tag: GEO Accession
attributes.value: GSM2769429