GSM3768332
GSM3768332: Fto OE rep3; Homo sapiens; OTHER
Total RNA was isolated using Trizol-LS. Poly(A) RNA was purified using Poly(A)Purist Mag kit (Thermofisher AM1922) according to manufacturer's instructions. Poly(A) RNA was fragmented using RNA fragmentation buffer (Ambion AM8740) and dephosphorylated at its 3' end with T4 PNK (NEB M0201) for 30min at 37oC. 5' phosphorylation was then initiated by adding 1mM ATP and incubating for 30min at 37oC. 3' ligation was then performed as previously described 2, where 5'-adenylated,3-dideoxyC DNA adapters were ligated with truncated T4 RNA ligase 2 (NEB M0242) in 1X ATP-free T4 RNA ligase buffer [50mM Tris pH 7.5, 60µg/ml BSA, 10mM MgCl2, 10mM DTT, 12.5% PEG8000] for 2hr at 25oC. 3'-ligated methylated RNA spike-in (Table S1) was added to ligated Poly(A) RNA and the mixture was denatured for 5min at 65oC before incubating for 2min on ice. This denatured RNA mixture was incubated overnight at 4oC with anti-m6A antibody (Synaptic Systems 202003) in 1X IP buffer [150mM NaCl, 10mM Tris pH 7.4, 0.1% IGEPAL CA-630 (Sigma I8896)] supplemented with RNasin Plus (Promega N2611). In parallel, Protein-A-dynabeads (Life Technologies 10002D) was blocked overnight at 4oC in 1X IP buffer supplemented with 0.5mg/ml BSA (Sigma A7906). The iced antibody-RNA mixture was crosslinked with 0.15J/cm2 254nm UV radiation six times. 1% of the antibody-RNA mixture was set aside as “input” and the rest (designated as “m6ACE”) was mixed with decanted BSA-blocked Protein-A-dynabeads for 1.5hr at 4oC. Beads bound with crosslinked RNA were then washed with the following cold buffers in this order: Wash buffer 1 [1M NaCl, 50mM HEPES-KOH pH 7.4, 1% Triton X-100, 0.1% Sodium Deoxycholate, 2mM EDTA], Wash buffer 2 [0.5M NaCl, 50mM HEPES-KOH pH 7.4, 1% IGEPAL, 0.1% Sodium Deoxycholate, 2mM EDTA], Wash buffer 3 [1% Sodium Deoxycholate, 25mM LiCl, 10mM Tris pH 8, 1% Triton X-100, 2mM EDTA], TE [10mM Tris pH 8, 1mM EDTA] and finally 10mM Tris pH 8. m6ACE RNA was subjected to 5' to 3' exonuclease treatment with XRN-1 (NEB M0338) for 1hr at 37oC. The m6ACE RNA-bead mixture was then washed with Wash buffer 1, Wash buffer 2, Wash buffer 3, TE and 10mM Tris pH 8. Both input and m6ACE RNAs were reverse crosslinked in elution buffer [1%SDS, 200mM NaCl, 25mM Tris pH 8, 2mM EDTA, 1mg/ml Proteinase K (Thermo Scientific EO0491)] for 1.5hr at 50oC. RNAs were ethanol-precipitated and ligated to 5'adapters (Table S1) with T4 RNA ligase (Ambion AM2140) for 16hr at 16oC. Reverse transcription primer (Table S1) was annealed (72oC 2min, ice 2min) and reverse transcription was performed with SuperscriptIII (Invitrogen 18080) for 1hr at 50oC, with the reaction stopped by incubating for 15min at 70oC. The cDNA was used for PCR amplification with Phusion High-fidelity PCR mastermix (NEB M0530) and Truseq PCR primers. Finally, primer-dimer and adapter-dimers were removed with AMpure XP beads before undergoing PE75 sequencing on the Illumina Nextseq platforms.
GEO
GSM3768332