Instrument: Illumina Genome Analyzer
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: The spleen and kidney tissues were disrupted in RLT buffer using a tissue lyser and RNA was extracted using the Qiagen RNeasy mini kit as per manufacturer’s protocol including the on-column DNase digestion. Blood RNA was isolated using Paxgene RNA isolation kit from Qiagen as per manufacturer’s instructions. Quality control of samples was done to determine RNA quantity and quality prior to their processing by RNA-seq. The concentration of total RNA samples was determined using NanoDrop 8000 (Thermo Scientific). The integrity of RNA samples was tested using Fragment Analyzer and High Sensitivity RNA kit (Advanced Analytical). 1ug of total RNA was input for library preparation using TruSeq RNA Sample Preparation Kit v2 (Illumina). Generated libraries were amplified with 8 cycles of PCR. Size of the libraries was confirmed using Fragment Analyzer and High Sensitivity DNA NGS kit (Advanced Analytical) and their concentration was determined by qPCR based method using Library quantification kit (KAPA). The libraries were first multiplexed (five per lane) and then sequenced on Illumina HiSeq2500 (Illumina) to generate 30 million of single end 50 base pair reads for each sample.