GSM6287566: Epididyaml primary adipocytes - young Crtc2 AKO_rep3; Mus musculus; RNA-Seq
Total RNA concentration was calculated by Quant-IT RiboGreen(Invitrogen, #R11490). To assess the integrity of the total RNA, samples are run on the TapeStation RNA screentape(Agilent). Only high-quality RNA preparations, with RIN greater than 7.0, were used for RNA library construction. A library was prepared with 1ug of total RNA for each sample by Illumina TruSeq mRNA Sample Prep kit (Illumina, Inc., San Diego, CA, USA). The first step in the workflow involves purifying the poly‐A containing mRNA molecules using poly‐T oligo‐attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using SuperScript II reverse transcriptase(Invitrogen) and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single 'A' base, and then ligation of the indexing adapters. The products are then purified and enriched with PCR to create the final cDNA library. The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and qualified using the TapeStation D1000 ScreenTape  (Agilent Technologies, Waldbronn, Germany). Indexed libraries were then sequenced using the HiSeq4000 platform (Illumina,San Diego, USA) by the Macrogen Incorporated.