GSM5766914: HepG2 WT rep2 (input); Homo sapiens; OTHER
GSM5766914
Three biological replicates of 40 million (2✕ 150 mm plates) HepG2 cells of stable overexpression of EGFP, METTL14 (WT), METTL14 (R298P) and METTL14 (D312A) were lysed by Trizol and the total RNA was extracted. The total RNA was treated by DNase I (5 U per 100 µg total RNA) and mRNAs were enriched by NEBNext Poly(A) mRNA magnetic isolation module (NEB E7490L). 8-10 µg of mRNA from each sample was fragmented to the size of ~120-nt by Ambion Fragmentation reagent (Thermo Scientific AM8740) in a proportional volume (10 µl reaction per 1 µg mRNA) at 70 ℃ for 7 min then precipitated in 75% ethanol with 10% volume of sodium acetate (3 M, pH 5.5) and GlycoBlue (1 µl, Invitrogen AM9515). The RNA fragments were dissolved in nuclease-free H2O at a concentration of 100 ng/µl, and 200 ng from each sample was spared as input. 5 µg of RNA fragment was denatured and incubated with 15 µg of anti-m6A antibody (Abcam ab151230) in immunoprecipitation (IP) buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA pH 8.0, 0.2 U/µl SUPERase·In) for 4 h at 4 ℃. The complex was incubated with 100 µl slurry of protein A/G beads blocked by BSA (0.5 mg/ml in IP buffer) at 4 ℃ overnight. The beads were washed by 0.9 ml, twice of each IP buffer, high-salt buffer (10 mM Tris pH 7.5, 1 M NaCl, 1% NP-40, 0.5% sodium deoxycholate), low-salt buffer (10 mM Tris pH 7.5, 50 mM NaCl, 0.1% NP-40), and FastAP buffer (10 mM Tris pH 8.0, 5 mM MgCl2, 100 mM KCl, 0.02% Triton X-100). The IP samples were dephosphorylated on-bead by 5 U of FastAP (Thermo Scientific EF0615) in 100 µl slurry at 37 ℃ for 30 min in a thermomixer with 15 s pulse-shaking at 1200 r.p.m. every 5 min, then washed by FastAP buffer and T4 RNA ligase buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT). A linker RNA oligonucleotides purchased from IDT was pre-adenylated at 5' -end by T4 RNA ligase I (NEB 0437) using a method described previously {Song, 2015 #6827}. The pre-adenylated linker RNA (100 pmol) was added to the 3' -end of IP RNAs on-bead by 500 U of T4 RNA ligase 2, truncated K227Q (NEB M0351) in 50 µl slurry (with a 25% final concentration of PEG8000) at 16 ℃ for an overnight-incubation in a thermomixer with pulse-shaking, then washed by T4 RNA ligase buffer and high-salt buffer. The input samples were dephosphorylated and ligated to the pre-adenylated linker RNA similarly in tubes, except the reactions were scaled-down 5-time and 2.5-time, respectively. Precipitation of RNA in 80% ethanol was performed after both reactions for the input samples. The IP RNA on-bead was eluted twice by incubation with 100 µg proteinase K in an 100 µl slurry (50 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA, 1% SDS, 0.4 U/µl SUPERase·In) at 50 ℃ for 1 h in a thermomixer with pulse-shaking then extracted by acid phenol-chloroform and precipitated in 75% ethanol with GlycoBlue and 0.1✕ volume of NaCl (2 M). Input and IP RNA were reversed transcribed by RT primer using SuperScript III and the excessive RT primer was removed by ExoSAP-IT. The RNA template in RT reactions was removed by NaOH (3 µl, 1 M) hydrolysis at 70 ℃ for 12 min and the reaction was neutralized by 3 µl HCl (1 M). The resulting first-strand was cleaned by MyONE Silane beads (Invitrogen 37002D) before ligated to an ssDNA adaptor fused with 10-random-nucleotide UMI (60 pmol) by T4 RNA ligase 1 (45 U) in a 30 µl reaction with 20% PEG8000, and cleaned again by MyONE Silane beads after ligation. The standard Nextera i5 and i7 barcodes were added to the adapted cDNA by a 13-cycle of PCR amplification. The dsDNAs were resolved on 2% agarose gel, and the fragments within the 175-250 bp range were eluted and pooled for Illumina NextSeq500 single-end 75-bp sequencing with expected sequencing depth of 25 million reads.