Illumina HiSeq 2000 paired end sequencing; Transcriptional analysis of murine kidneys after caloric restriction and hypoxic preconditioning in an ischemia-reperfusion injury model
Transcriptional analysis of murine kidneys after caloric restriction and hypoxic preconditioning in an ischemia-reperfusion injury model
Mice kidneys were snap-frozen after nephrectomy. RNeasy mini Kit (Qiagen, Hilden, Germany) was used to isolate RNA from snap-frozen kidneys as per manufacturer's instructions. Ribosomal RNA was removed by using biotinylated target-specific oligos combined with Ribo-Zero rRNA removal beads from 1µg totalRNA input. The Ribo-Zero Human/Mouse/Rat kit depletes samples of cytoplasmic rRNA. Following purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single'A' base and subsequent ligation of the adapter. The products are purified and enriched with PCR (20ul template, 15cycles) to create the final cDNA library. After library validation and quantification, equimolar amounts of library were pooled. Pools of 5-6 libraries each were quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System and sequenced on Hiseq 2000.