m6A-seq
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quality and quantity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Approximately more than 200 ug of total RNA was subjected to isolate Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). Following purification, the poly(A) mRNA fractions is fragmented into ~100-nt-long oligonucleotides using divalent cations under elevated temperature. Then the cleaved RNA fragments were subjected to incubated for 2h at 4 with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50mM Tris-HCl, 750mM NaCl and 0.5% Igepal CA-630) supplemented with BSA (0.5gl1). The mixture was then incubated with protein-A beads and eluted with elution buffer (1  IP buffer and 6.7mM m6A). Eluted RNA was precipitated by 75% ethanol. Eluted m6A-containing fragments (IP) and untreated input control fragments are converted to final cDNA library in accordance with a strand-specific library preparation by dUTP method. The average insert size for the paired-end libraries was ~10050 bp. And then we performed the paired-end 2150bp sequencing on an Illumina Novaseq 6000 platform at the LC-BIO Bio-tech ltd (Hangzhou, China) following the vendor's recommended protocol.
PFIM60_20_2_IP