GSM2701993: Untreated rep2; Mus musculus; RNA-Seq
RNA was isolated from the retinas with the RNeasy kit (Qiagen, Hilden, Germany). Library preparation and RNAseq were carried out as described in the Illumina TruSeq Stranded mRNA Sample Preparation Guide, the Illumina HiSeq 1000 System User Guide (Illumina, Inc., San Diego, CA, USA), and the KAPA Library Quantification Kit - Illumina/ABI Prism User Guide (Kapa Biosystems, Inc., Woburn, MA, USA). In brief, 300 ng of total RNA was used for purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented to an average insert size of 200-400 bases using divalent cations under elevated temperature (94 °C for 4 min). 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 in the Second Strand Marking Mix (SMM), followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The incorporation of dUTP in second strand synthesis quenches the second strand during amplification, because the polymerase used in the assay is not incorporated past this nucleotide. The addition of Actinomycin D to First Strand Synthesis Act D mix (FSA) prevents spurious DNA-dependent synthesis, while allowing RNA-dependent synthesis, improving strand specificity. These cDNA fragments then had the addition of a single 'A' base and subsequent ligation of the adapter. The products were purified and enriched with PCR to create the final cDNA library. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA). Equimolar amounts of each library were pooled, and the pools were used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on an HiSeq 1000 instrument using the indexed, 50 cycles single-read (SR) protocol and the TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide. Image analysis and base calling resulted in .bcl files, which were converted into .fastq files with the CASAVA1.8.2 software.