GSM6774558: IgG-control_3; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol Reagent (Invitrogen). After the recovery of the aqueous phase using chlorophorm, a DNAse treatment was performed using the Mini RNeasy kit (Qiagen) following manufacturer instructions.  cDNA libraries were generated using the Illumina TruSeq RNA Sample Preparation Kit, and sequenced on a Illumina HiSeq2500 sequencer. Total RNA samples (1µg) were used. Average sample RNA Integrity Number was 9.8 (range 9.6-10) when assayed on an Agilent 2100 Bioanalyzer. PolyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters with "NEBNext Ultra II Directional RNA Library Prep Kit for Illumina" (NEB, Cat. No. E7760) as recommended by the manufacturer. This kit incorporates dUTP during 2nd strand cDNA synthesis, which implies that only the cDNA strand generated during 1st strand synthesis is eventually sequenced. Adapter-ligated library was completed by PCR with Illumina PE primers. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced on an Illumina NextSeq 500 by following manufacturer's protocols.