Total RNA samples from INS-1 832/13 cells were tested for purity and integrity verification (NanoDrop Spectrophotometer and Agilent 2100 BioAnalyzer, respectively) in preparation for RNA-seq library preparation. Each library was generated using Illumina's “TruSeq RNA Sample Preparation v2 Guide” (Illumina Part no. 15026495, Rev. C) and Illumina's TruSeq RNA Sample Prep Kit v2 (Illumina Inc., San Diego, CA). mRNA was purified from 1 μg of total RNA using poly-T oligo-attached magnetic beads. Subsequently, each mRNA sample was fragmented into small pieces using divalent cations under elevated temperature, and double-stranded cDNAs were synthesized using SuperScript II (Invitrogen) and random primers for first strand cDNA synthesis followed by second strand synthesis using DNA polymerase I and RNase H for removal of mRNA. Double-stranded cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter). cDNAs were end-repaired by T4 DNA polymerase and Klenow DNA polymerase and phosphorylated using T4 polynucleotide kinase. The blunt-ended cDNA was purified using Agencourt AMPure XP beads. The cDNA products were incubated with Klenow DNA polymerase to add an “A” base (adenine) to the 3′-end of the blunt phosphorylated DNA fragments. DNA fragments were ligated to Illumina adapters, which have a single “T” base (thymine) overhang at their 3′-end. The adapter-ligated products were purified using Agencourt AMPure XP beads. Adapter-ligated DNA was amplified in a linker-mediated PCR for 11 cycles using PhusionTM DNA polymerase and Illumina's PE genomic DNA primer set and then purified using Agencourt AMPure XP beads.
The quality and quantity of the finished libraries were assessed using an Agilent DNA 1000 series chip assay and QuantIT PicoGreen dsDNA kit (Invitrogen), respectively, and libraries were standardized to 2 μm. Cluster generation was performed using standard Cluster Kits (version 3) and the Illumina Cluster Station. Paired end 100-bp sequencing was performed using standard SBS chemistry (version 3) on an Illumina HiSeq2000 sequencer at the University of Wisconsin Biotechnology Center (Madison, WI). Images were analyzed using the standard Illumina Pipeline, version 1.8.2. The average number of reads/sample was 50 million. Results were analyzed by the Bioinformatics staff of the University of Wisconsin Biotechnology Center. Full paper
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