Heat labile udg enzyme

Total RNA of each sample was extracted and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by fragmentation using Magnesium RNA Fragmentation Module (NEB, USA), and synthesis of U-labeled second-stranded DNAs using SuperScript™ II Reverse Transcriptase (Invitrogen, USA), E. coli DNA polymerase I (NEB, USA), RNase H (NEB, USA) and dUTP Solution (Thermo Fisher, USA). After single- or dual-index adapters were ligated to the fragments, the U-labeled second-stranded DNAs were treated with heat-labile UDG enzyme (NEB, USA), and the ligated products were amplified with PCR. Sequencing data were generated on Illumina Novaseq™ 6000 (LC Sciences, USA) with 2 × 150 bp paired-end sequencing (PE150) following the vendor's recommended manual.

The total RNA was fragmented and then incubated with m⁶A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris–HCl, 750 mM NaCl, and 0.5% Igepal CA-630) for 2 h at 4°C. Then, the IP RNA was reverse-transcribed to cDNA, and U-labeled second-stranded DNAs were synthesized using cDNA with E. coli DNA polymerase I, RNase H (NEB, United States), and dUTP Solution (Thermo Fisher Scientific, United States). Then, the strands were prepared with A-base and ligated to the indexed adapters containing a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. AMPureXP beads were used to screen fragments of the right size. The U-labeled second-stranded DNAs treated with heat-labile UDG enzyme (NEB, United States) were next amplified by PCR to generate a cDNA library with an average insert size of 300 ± 50 bp. Finally, 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq^TM 6000 (LC-BioTechnology Co., Ltd., Hangzhou, China), following the manufacturer’s recommended protocol.