OE1_2 (CRX859381)
RNA from total samples was isolated and purified using TRIzol according to the protocol provided by the manufacturer. The amount and purity of total RNA was then quality controlled with a NanoDrop ND-1000 and the integrity of the RNA was examined by a Bioanalyzer 2100. total RNA >1 ug were sufficient for downstream experiments. The mRNA with PolyA (polyadenylate) was specifically captured by two rounds of purification using oligo(dT) magnetic beads. The captured mRNAs were fragmented using the Magnesium Ion Interrupter Kitunder high temperature conditions, 94 degreeCC, 5-7 min. The fragmented RNA was passed through reverse transcriptase to synthesize cDNA. cDNA was then synthesized using E. coli DNA polymerase I with RNase H and RNase H for two-stranded synthesis to convert these composite double strands of DNA and RNA into DNA duplexes, while the duplexes were doped with dUTP Solution to make up the ends of the double-stranded DNA to flat ends, and then An A base was added to each of its ends to enable it to be ligated to a junction with a T base at the end, and its fragment size was screened and purified using magnetic beads. The second strand was digested with UDG enzyme, and the fragment size of 300 bp +/- 50 bp was formed by PCR-pre-denaturation holding at 95 degreeCC for 3 min, denaturation at 98 degreeCC for a total of 8 cycles of 15 s each, annealing to 60 degreeCC holding at 60 degreeCC for 15 s, extension at 72 degreeCC for 30 s, and a final extension held at 72 degreeCC for 5 min. 50bp library (strand-specific library). Finally, we sequenced it bipartite using illumina NovaseqTM 6000 following the standard operation with sequencing mode PE150.