GSM5706038: Hippocampus WT rep1; Mus musculus; RNA-Seq
Hippocampi were extracted, flash frozen on dry ice, and RNA was harvested using RNAEasy Mini kit (Qiagen). 500 ng of total RNA was used for poly(A)-mRNA selection using Oligo (dT) magnetic beads and subsequently fragmented to approximately 300bp. cDNA was synthesized using reverse transcriptase (SuperScript II, Invitrogen) and random primers. The second strand of the cDNA incorporated dUTP in place of dTTP. Libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit v2 according to the manufacturer's protocol. Briefly, double-stranded DNA was further used for library preparation. dsDNA was subjected to A-tailing and ligation of the barcoded Truseq adapters. All purification steps were performed using AMPure XP Beads. Library amplification was performed with PCR using the primer cocktail supplied in the kit. Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity, check the size distribution, and then quantified by qPCR using the KAPA Library Quantification Kit (KapaBiosystems, Merck, Darmstadt, Germany) before amplification Illumina's cBot. Libraries were sequenced 1 x 50bp on Illumina's HiSeq 2500.