Paired end solexa adaptors

Total RNA of each sample was isolated using a Quick RNA isolation kit (Bioteke Corporation, Beijing, China) and then characterized on a 1% agarose gel and examined with a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The RIN (RNA integrity number) values (>8.0) of these samples were assessed using an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). The construction of the libraries and the RNA-Seq were performed by the Biomarker Biotechnology Corporation (Beijing, China). mRNA was enriched and purified with oligo(dT)-rich magnetic beads and then broken into short fragments. Taking these cleaved mRNA fragments as templates, first- and second-strand cDNA were synthesized. The resulting cDNAs were then subjected to end-repair and phosphorylation using T4 DNA polymerase and Klenow DNA polymerase. After that, an ‘A’ base was inserted as an overhang at the 3′ ends of the repaired cDNA fragments and Illumina paired-end solexa adaptors were subsequently ligated to these cDNA fragments to distinguish the different sequencing samples. To select a size range of templates for downstream enrichment, the products of the ligation reaction were purified and selected on a 2% agarose gel. Next, PCR amplification was performed to enrich the purified cDNA template. Finally, the four libraries were sequenced using an Illumina HiSeq™ 2000.

The construction of the libraries and the RNA-Seq were performed by the Biomarker Biotechnology Corporation (Beijing, China). mRNA was enriched and purified with oligo(dT)-rich magnetic beads and then broken into short fragments. Using these cleaved mRNA fragments as templates, first- and second-strand cDNA were synthesized. The resulting cDNAs were then subjected to end-repair and phosphorylation using T4 DNA polymerase and Klenow DNA polymerase. Subsequently, an ‘A’ base was inserted as an overhang at the 3′ ends of the repaired cDNA fragments, and Illumina paired-end solexa adaptors were subsequently ligated to these cDNA fragments to distinguish the different sequencing samples. The products of the ligation reaction were purified and selected on 2% agarose gel for downstream enrichment. Then the purified cDNA template was enriched by PCR amplification. Finally, 6 libraries were sequenced using an Illumina HiSeq™ 2500 (SRA accession number: SRP112400). Data analysis and base calling were performed using the Illumina instrument software.