GSM8987270: m1G precipitated RNA, WT control, experiment 2, replicate 3; Homo sapiens; RIP-Seq
5-10μg polyA+ enriched RNA of control and TRMT5 KD transfected PANC-1 cells was fragmented to 100-200nt average size using RNA fragmentation reagents following the manufacturer's instructions (ThermoFisher Scientific). The fragment sizes were confirmed using an Agilent Tapestation 2200 with RNA screentape (Agilent).  Fragmented RNA was diluted with RNAse free water to 755μl and heated at 75ºC for 5 minutes to disrupt secondary structures, before being immediately placed on ice for >1 minute. 200U RNAseOUT (Life Technologies), 10μg anti-m1G antibody (ab208199) or GFP antibody and 200μl 5X IP buffer (50mM Tris-HCl pH7.4, 750mM NaCl, 0.5% Igepal CA-630) were then mixed with the RNA in nonstick, RNase-free tubes (ThermoFisher Scientific). The reaction was incubated overnight at 4ºC on a rotating mixer to allow the antibody to bind to the modification of interest. Protein A Dynabeads (Life Technologies) and Protein G Dynabeads (Life Technologies Ltd) were combined at a 1:1 ratio (allowing for 50μl per reaction) and washed with 1X IP buffer (10mM Tris-HCl pH7.4, 150mM, 0.1% IGEPAL CA-630) and then resuspended in 1ml 1X IP buffer containing 0.5mg/ml bovine serum albumin (BSA) to block non-specific binding to the beads. The beads were then incubated for 2 hours at 4ºC with rotation. The beads were then washed three times with 1X IP buffer and resuspended in the original volume. 50μl of blocked beads were used for each IP reaction. The pre-blocked beads were then added directly to the fragmented RNA/antibody complex prepared previously. The reactions were then incubated for 2 hours at 4ºC with rotation. The following wash steps were then used to reduce non-specific binding: low salt buffer (10mM Tris pH7.4, 75mM NaCl, 0.1% IGEPAL CA-630), high salt buffer (10mM Tris pH7.4, 200mM NaCl, 0.1% IGEPAL CA-630) and TEN buffer (10mM Tris-HCl pH7.4, 1mM EDTA, 0.05% IGEPAL CA-630). The immunoprecipitated RNA was eluted by competition with free modified nucleoside. Each IP reaction was eluted with 450μl competition elution buffer (90μl 1X IP buffer, 6mM free nucleoside, 7μl RNAseOUT and RNAse free water up to 450μl), then incubated at 4ºC for 2h with shaking. Eluted RNA was recovered using Trizol LS reagent (ThermoFisher Scientific), followed by isopropanol precipitation and precipitated overnight at -80ºC. The immunoprecipitated RNA was used for sequencing using NEXTflex Rapid Directional sequencing kit (PerkinElmer), adjusting the ratio of AMPure XP beads (Beckman Coulter) for the desired size selection.