GSM5494754: RNA-seq_635_SCA7_KI_homo_Rep3; Mus musculus; RNA-Seq
Retinas were flash frozen in liquid nitrogen and stored at -800C. The total RNA was obtained using RNeasy Micro Kit (Qiagen). Total RNA-Seq libraries were generated from 1 µg of total RNA using TruSeq Stranded Total RNA Library Prep Gold kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, cytoplasmic and mitochondrial ribosomal RNA (rRNA) was removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the remaining RNA was fragmented into small pieces using divalent cations at 94°C for 2 minutes. RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (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). Surplus PCR primers were removed using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were quality controlled and quantified using capillary electrophoresis.