cDNA Library Construction

Poly (A) RNA was purified from total RNA (10 μg) using poly T oligo attached magnetic beads using two rounds of purification. Then the poly (A) RNA was fragmented into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were reverse transcribed to create the cDNA, which were next used to synthesize U labeled second stranded DNAs with Escherichia coli DNA polymerase I, RNase H and dUTP. An A base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T base overhang for ligating the adapter to the A tailed fragmented DNA. Single or dual index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat labile UDG enzyme treatment of the U labeled second stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95°C for 3 min; 8 cycles of denaturation at 98°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s; and then final extension at 72°C for 5 min. The average insert size for the final cDNA library was 300 bp (±50 bp). At last, we performed the 150 bp paired end sequencing on an Illumina Hiseq 6000 (LC Bio, China) following the vendor’s recommended protocol.