title: RNA-Seq of normal human liver tissue - Illumina HiSeq, specimen 5
design: Total RNA was isolated using Extract RNA kit (Eurogene, Russia). RNA integrity was evaluated using both capillary electrophoresis by Bioanalyzer 2100 System (Agilent Technologies, USA) and agarose electrophoresis. RIN numbers varied from 7.3 to 9.1. Next, we synthesized full-length-enriched double stranded cDNA using Mint-2 kit (Eurogene, Russia). Briefly, oligo(dT) primers were annealed to poly(A) 3-tails of RNA. When Mint reverse transcriptase reaches the 5-end of the mRNA, it adds several non-template nucleotides, primarily deoxycytidines, to the 3-end of the newly synthesized first-strand cDNA. This enables annealing oligo(gG) primers (PlugOligo) to the 5-tails and synthesis of the second cDNA strand. Next, we prepared sequencing-ready cDNA libraries using Qiaseq FX DNA Library Kit (Qiagen, USA) according to the manufacturers protocol. Library quality control was carried out using Bioanalyzer 2100 System (Agilent Technologies, USA) in order to evaluate insert distribution. Clustering and sequencing was carried out using Illumina HiSeq 2500 system (2 lanes per 8 samples) according to the manufacturers protocols (Denature and Dilute Libraries Guide; Sequencing in Rapid Run Mode).