Fast 2000 kit

Total RNA was extracted from the cells using fast2000 kit (Fast Agen) and RNA was treated with DNase I (1U/µl, Thermo Fisher Scientific, USA). Magnetic beads with Oligo (dT) were used to enrich mRNA. Fragmentation buffer was used to obtain short fragments of mRNA (17 (link)). cDNA was then synthesized using the mRNA fragments as templates with the Agencourt AMPure XP Kit (Beckman Coulter, Inc.), according to the manufacturer's protocol. Short fragments were purified and recycled with EB buffer for end reparation and base ‘A' addition (17 (link)). Subsequently, the 200-500 bp size of the fragments was selected for polymerase chain reaction (PCR) amplification as follows: 98°C for 30 sec, one cycle; 98°C for 10 sec, 15 cycles; 60°C for 30 sec, 72°C for 30 sec and last cycle at 72°C for 5 min. During the quality control (QC) steps, the constructed library was qualified and quantified by Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) and ABI StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) quality inspection. Finally, Illumina HiSeq™ 2000 (Illumina, Inc.) was used for sequencing (Beijing Genomics Institute).