GSM5917906: scc25, IP; Homo sapiens; RIP-Seq
m6A MeRIP is based on the previously described m6A-seq protocol[1] with several modifications: 30 μl of protein A magnetic beads (10002D; Thermo Fisher Scientific) and 30 μl of protein G magnetic beads (10004D; Thermo Fisher Scientific) were washed twice by IP buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O), resuspended in 500 μl of IP buffer, and tumbled with 5 μg anti-m6A antibody at 4 C for at least 6 hours. Following 2 washes in IP buffer, the antibody-bead mixture was resuspended in 500 μl of the IP reaction mixture containing fragmented total RNA, 100 μl of 5× IP buffer, and 5 μl of RNasin Plus RNase Inhibitor (N2611; Promega, Madison, WI) and incubated for 2 hours at 4 C. In the low/high salt-washing method, the RNA reaction mixture was washed twice in 1,000 μl of IP buffer, twice in 1,000 μl of low-salt IP buffer (50 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O), and twice in 1,000 μl of high-salt IP buffer (500 mM NaCl, 10 mM Tris-HCl [pH 7.5],0.1% IGEPAL CA-630 in nuclease-free H2O) for 10 minutes each at 4 C. After extensive washing, the m6Aenriched fragmented. RNA was eluted from the beads in 200 μl of RLT buffer supplied in RNeasy Mini Kit (74106; QIAGEN; Germany) for 2 minutes at room temperature. A magnetic separation rack was used to pull beads to the side of the tube. Supernatant was collected to a new tube, and 400 μl of 100% ethanol was added to it. The mixture was transferred to an RNeasy MiniElute spin column (QIAGEN) and centrifuged at >12,000 rpm at 4 C for 1 minute. The spin column membrane was washed with 500 μl of RPE buffer once, then 500 μl of 80% ethanol once, and centrifuged at full speed for 5 minutes at 4 C to remove the residual ethanol. The m6A-enriched RNA was eluted with 14 μl ultrapure H2O. The amount of 2 μl of 14 μl eluted RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Once successfully immunoprecipitated methylated RNA was confirmed, further transcriptome-wide interrogation was pursued by deep sequencing using SMARTer Stranded Total RNA-Seq Kit version 2 (Pico Input Mammalian; 634413; Takara/Clontech, Japan) according to the manufacturer's protocol. A purified library was quantified using a Qubit Fluorometer (Thermo Fisher Scientific), and the size distribution was checked using the Bioanalyzer 2100 system (Agilent Technologies). The library preparations were sequenced on an Illumina Novaseq 6000 platform and 150 bp paired-end reads were generated. MeRIP-seq