Illumina HiSeq 2000 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. miRNA libraries were prepared using the Illumina TruSeq Small RNA Sample Preparation Kit. Library preparation started with 1 µg of total RNA. After ligation with 3 and 5 RNA adaptors, RT–PCR was used for first-strand synthesis. Fragments were amplified to complete adaptor constructs and gel purified for size selection. After library validation and quantification (using an Agilent 2100 Bioanalyzer), equimolar amounts of the libraries were pooled. Pools were quantified using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System and sequenced on Hiseq 2000.