GSM3839970: sgRNA_FTO_mutation_m6A_seq; Homo sapiens; OTHER
Total RNA was isolated using Trizol reagent followed by fragmentation using freshly prepared RNA fragmentation buffer (10 mM Tris-HCl pH 7.0, 10 mM ZnCl2). 180 μg fragmented RNA was incubated with 6 μg anti-m6A antibody in 1× m6A IP buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.1% Igepal CA-630) supplemented with 6 µL RNase OUT (40 U/µL) and 2 mM RVC for 2 h at 4°C. The m6A-IP mixture was then incubated with Protein A/G beads and rotated for additional 3 h at 4°C. Following four washes in 1× IP buffer, m6A-containing RNA fragments were eluted by incubation with 200 μl elution buffer (6.7 mM N6-methyladenosine 5'-monophosphate sodium salt in 1× IP buffer) for 1 h at 4°C. Eluted RNA was used for libraries constrution. m6A-containing RNAs (m6A-seq) or fragmented total RNAs (RNA-seq) were dephosphorylated for 1 hr at 37°C in 15 µl reaction (1× T4 polynucleotide kinase buffer, 10 U SUPERase_In and 20 U T4 polynucleotide kinase). The products were separated on a 15% polyacrylamide TBE-urea gel (Invitrogen) and visualized using SYBR Gold (Invitrogen). The gel with selected regions 40-60 nt was excised and disrupted by using centrifugation through the holes at the bottom of the tube. RNA fragments were dissolved by soaking overnight in 400 μl gel elution buffer (300 mM NaOAc, pH 5.5, 1 mM EDTA, 0.1 U/ml SUPERase_In). The gel debris was removed using a Spin-X column (Corning), followed by ethanol precipitation. Purified RNA fragments were re-suspended in nuclease-free water. Poly-(A) tailing reaction was carried out for 45 min at 37°C (1 × poly-(A) polymerase buffer, 1 mM ATP, 0.75 U/μl SUPERase_ In and 3 U E. coli poly-(A) polymerase).
GEO Accession
GSM3839970