Comparative sequence analyses of genome and transcriptome reveal novel transcripts and variants in the Asian elephant Elephas maximus
Total RNA from lymphocytes was isolated from peripheral blood (PBMCs) using Trizol and the quality of RNA was analyzed using Bioanalyzer 2100 RNA 6000 nano Chip (Agilent).One μg RNA samples with RIN value of 7.2 were used for library preparation. RNA-seq library was prepared using TruSeq RNA Sample Prep Kits (Illumina). Briefly, poly-A containing mRNA molecules were enriched using poly-T oligo-attached magnetic beads. mRNA was fragmented to obtain desired size using divalent cations under elevated temperature. The cleaved RNA fragments were used to synthesize 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 were end repaired, and adapters were ligated. The library was further enriched with PCR to create the final cDNA library.