The total RNA was extracted using the TrIzol reagent (Life Technologies, Beijing, China) according to the manufacturer’s protocols. Agarose gel electrophoresis and Agilent 2100 (Agilent Technologies, CA, USA) were applied to assessed RNA integrities , while Nanodrop (IMPLEN, CA, USA) and Qubit (Life Technologies, CA, USA) were used for concentration detection. Subsequently, RNA from 4 individuals were used for cDNA library construction and Illumina deep sequencing (NEB, USA) respectively. Briefly, the poly(A) RNA enrichment by oligo(dT) magnetic beads from total RNA, then fragment as template and reverse transcription with random hexamer primers to synthesis first-strand cDNA. Following supplement with buffer, dNTPs, DNA polymerase I and RNase H to obtain second-strand cDNA. Double-stranded cDNA was purified by AMPure XP beads (Beckman Coulter, Beverly, USA). Subsequently, it would undergo end-repairment, an A tail added, ligation to paired-end Illumina sequencing adaptors, selection size of fragment by AMPure XP beads and amplification by polymerase chain reaction (PCR) for lirary preparation. The high quanlity library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated.
GEO Accession
GSM2522181