GSM8331752: CMV_2b_1_IP; Arabidopsis thaliana; OTHER
Total RNA was isolated and purified using TRIzol reagent, and Poly (A) RNA is purified from 50μg total RNA using Dynabeads Oligo (dT) using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module. The cleaved RNA fragments were used for IP with m6A-specific antibody, and the resulting IP product was used for library construction. Libraries were prepared according to Illumina's Protocol. Briefly, DNA was end-repaired using a combination of E. coli DNA polymerase I, RNase H and dUTP Solution. 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. The average insert size for the final cDNA library was 300±50 bp.At last, we performed the 2×150bp paired-end sequencing (PE150) on an illumina Novaseq™ 6000 following the vendor's recommended protocol.