NextSeq 500 sequencing; Integrative Omics Reveals Subtle Molecular Perturbations Following Ischemic Conditioning in a Porcine Kidney Transplant Model
Integrative Omics Reveals Subtle Molecular Perturbations Following Ischemic Conditioning in a Porcine Kidney Transplant Model
Cycles of ischemia were performed on the kidney before explantation, by clamping the distal aorta. Sixteen tissue samples from the kidney upper pole (weighing approximately 20 mg) were lysed using a Precellys 24 homogeniser (Bertin Instruments) in 350 µL of RNeasy Mini kit RLT lysis buffer.  RNA was then extracted using RNeasy Mini kit (Qiagen, cat number: 74104) as per manufacturer's instructions. Purified RNA was eluted in 40 µL RNase-free H2O and the approximate concentration evaluated using a NanoDrop ND-1000 spectrophotometer. Approximately, 450 ng of purified RNA was converted to cDNA by using high-capacity reverse transcription kit (Applied Biosystems, cat number: 4368814). For each reaction, a mix of 2 µL 10x RT buffer, 0.8 µL 25x dNPT mix, 2 µL 10x RT random primers, 1 µL Multiscribe reverse transcriptase, 3.2 µL nuclease free H2O and 11 µL of RNA (40.9 ng/µL) was prepared. This was then primed at 25°C for 10 min, incubated at 37°C for 120 min and inactivated at 85°C for 5 min in a thermal cycler (VWR UNO 96). cDNA was then stored at -20°C until further use.  FASTQ files were converted to Binary-sequence Alignment Format (BAM) files using HISAT2 (v2.1.0) and Samtools (v1.3).