Total RNA was extracted from cells using Nucleospin® RNA kit (Macherey Nagel, Cat# 740955) For sequencing, RNA libraries were prepared using standard Illumina protocols. Total RNA sample is digested by DNaseІ (NEB), and purified by oligo-dT beads (Dynabeads  mRNA purification kit, Invitrogen), then poly (A)-containing mRNA were fragmented into 130bp  with First Strand buffer. First-strand cDNA is generated by N6 primer, First Strand Master Mix and  Super Script II reverse transcription(Invitrogen) (Reaction condition：25℃ for 10 min；42℃ for 40  min；70℃for 15 min). Then add Second Strand Master Mix to synthesize the second-strand cDNA  (16℃ for 1h). Purified the cDNA with Ampure XP Beads (AGENCOURT), then combine with End  Repair Mix, incubate at 20℃for 30min. Purified and add A-Tailing Mix, incubate at 37℃ for 30  min. Then combine the Adenylate 3'Ends DNA, Adapter and Ligation Mix, incubate the ligate  reaction at 20℃for 20 min. Several rounds of PCR amplification with PCR Primer Cocktail and  PCR Master Mix were performed to enrich the cDNA fragments. Then the PCR products are  purified with Ampure XP Beads (AGENCOURT).
GEO Accession
GSM3656292