HCC2
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad CA, USA) according to the manufacturer's protocol. Approximately 5 ug of total RNA was used to deplete ribosomal RNA according to the manuscript of the Ribo-ZeroTM rRNA Removal Kit (Illumina, San Diego, USA). After removing ribosomal RNAs, the left RNAs were 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 synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP. An A-base is 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 are 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 are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300 bp (±50 bp). At last, we performed the paired-end sequencing on an Illumina Hiseq 4000 (LC Bio, China) following the vendor's recommended protocol.