GSM5268421: GM_input_Rep3; Bos taurus; RNA-Seq
Total RNA was extracted using RNAiso reagent (Takara, Daling, China). The quality and quantity of the total RNA were analyzed by Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN > 7.0. Poly (A) mRNA was isolated from total RNA over 200 ug using poly-T oligo attached magnetic beads (Invitrogen, Massachusetts, USA). Following purification, the poly(A) mRNA fractions are fragmented into about 100-nt-long oligonucleotides by divalent cations at high temperatures. The cleaved RNA fragments were then incubated with m6A-specific antibody (202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630) supplemented with BSA (0.5 μg μl−1) at 4 °C for 2h. Then the mixture was incubated with protein-A beads and eluted with elution buffer (1 × IP buffer and 6.7mM m6A). The eluted RNA was precipitated with 75% ethanol. According to the strand-specific library prepared by dUTP method, the eluted fragments containing m6A (IP) and the untreated input control fragments were converted to final cDNA library. The average insert size for the paired terminal libraries was ~100 ± 50 bp. And then we performed the paired-end 2×150bp sequencing on an Illumina Novaseq™ 6000 platform at the LC-BIO Bio-tech ltd (Hangzhou, China) following the vendor's recommended protocol.