title: GSM4928943: C _RT_655_F_27; Coturnix japonica; RNA-Seq
library_construction_protocol: RNA extraction was performed using All Prep RNA/DNA Mini kit (Qiagen), according to the manufacturer's instruction. RNA concentration was obtained on a Nanodrop ND-1000 UV-Vis Spectrophotometer. RNA integrity was verified by on an Agilent 2100 bioanalyzer with a RNA Nano kit (Agilent), according to the manufacturer's instruction. All sample RIN was above 8.5. RNA-Seq libraries were generated from 500ng of total RNA using TruSeq Stranded mRNA Library Prep Kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, CA), according to manufacturer's instructions. 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. Strand specificity was achieved by replacing dTTP with dUTP during second strand cDNA synthesis 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 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC) to create the cDNA library. Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.
attributes.tag: GEO Accession
attributes.value: GSM4928943