Congen_2 (CRX1872838; CRA028275)
After total RNA was extracted, mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module under 94oC 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript II Reverse Transcriptase, which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP Solution. An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. 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 95oC for 3 min; 8 cycles of denaturation at 98oC for 15 sec, annealing at 60oC for 15 sec, and extension at 72oC for 30 sec; and then final extension at 72oC for 5 min. At last, we performed the 2 X150bp paired-end sequencing (PE150) on an Illumina Novaseq 6000.