Rabbit igg control bound to protein a dynabeads

Biological replicates of control and TARBP2 knockdown cells (MDA-LM2 background) were collected and processed as previously described (Alarcón et al., 2015b (link)). 1×10⁷ cells per sample were lysed using LB1 buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% Triton X-100) and 1X protease inhibitor cocktail (Thermo Scientific). The nuclear fraction was then lysed with M-PER buffer (Thermo Scientific) and diluted tenfold in dilution buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl) before the immunoprecipitation. Rabbit anti-m6A antibody (Synaptic Systems) and rabbit IgG control bound to protein A dynabeads (Invitrogen) were used for the immunoprecipitations. The immunoprecipitated RNA was eluted with N6-methyladenosine (Sigma-Aldrich), ethanol precipitated and resuspended in water. RNA was barcoded using ScriptSeq V2 kit (Epicentre) and sequenced at Rockefeller University Genomics Core.

3×10⁷ cells/sample were lysed using LB1 buffer (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton x-100, and protease inhibitors). The nuclear fraction was then lysed with M-PER buffer (Thermo Scientific) and diluted 10-fold in dilution buffer (50mM Tris-Cl, pH 7.4, 100mM NaCl) before the immunoprecipitation. Rabbit α-m6A antibody (Synaptic Systems) and rabbit IgG control bound to protein A Dynabeads (Invitrogen) were used for the immunoprecipitations. The immunoprecipitated RNA was eluted with N6-methyladenosine (Sigma-Aldrich), ethanol precipitated and resuspended in water. RNA was barcoded using ScriptSeq V2 kit (Epicentre) and sent for sequencing.