D2LH
Total RNAs were enriched by magnetic beads with Oligo (dT) and interrupted into short segments with the addition of a fragmentation buffer after samples were qualified. Subsequently, double-stranded cDNA was synthesized with M-MuLV Reverse Transcriptase (RNase H-) and DNA Polymerase I and RNaseH, subjected to terminal reparation, and serial sequencing. The library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA) in order to select cDNA fragments of preferentially 150~200 bp in length. Selected double-stranded cDNA was subjected to PCR enrichment, construction, and sequencing of the RNA-seq library for each sample was conducted (Compass Biotechnology, Beijing, China) based on the protocols of Illumina HiSeqTM2500/MiSeq to generate paired-end reads (125bp/157 bp in length).