Transcriptome library preparation and RNA-seq

Total RNA was extracted using the RNeasy® Mini kit according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Total RNA was used to generate the complementary DNA (cDNA) libraries for paired-end sequencing using mRNA-seq Sample Preparation Kit (Illumina). Briefly, 4 μg of total RNA from each sample was used for polyA mRNA selection using polyT oligo-conjugated magnetic beads by two rounds of purification. The cleaved mRNA fragments were reverse transcribed and then converted into double-strand cDNA. Following end repair and A tailing, adapters complementary to sequencing primers were ligated to the ends of DNA fragment. Finally, the ligation products were further purified on 2% agarose gels and 200–250 basepair (bp) fragments were selected for downstream enrichment by 15 cycles of PCR followed by purification using QIAquick PCR purification kit (Qiagen). The libraries were sequenced and quantified with an Illumina HiSeq™ 2000 system.