GSM8743494: Mettl3 KO MTL3 eclip-1; Mus musculus; OTHER
UV crosslinked WT and Mettl3 KO Rx:GFP RPCs were collected and resuspended in 600µl of RIPA lysis buffer with freshly added Protease Inhibitor Cocktail (Roche, 11873580001) and 1 μl of Superase-In RNase inhibitor (Thermo Fisher, AM2694)) for 15 min on ice and then sonicated with Qsonica 800R (30 s, on; 30 s off; 20 minutes, 30% amplitude). Sonicated lysates were centrifuged at 13,000 × g for 15 min at 4 °C. Clarified lysates were incubated with 15µl METTL3 antibody (Abcam, Ab195352) at 4 °C overnight. Two percent of lysate was saved as size-matched input. 30µl of Protein A Dynabeads (Thermo Fisher, 10001D) were added into IP tubes and incubated at 4 °C for 2 hrs. The immunoprecipitated (IP) samples were washed with low salt buffer (20 mM Tris-HCl pH 8, 150mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, freshly added RNase Inhibitor at 1:1000 dilution) twice and then washed with LiCl wash buffer (10 mM Tris-HCl pH 8, 0.25 M LiCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, freshly added RNase Inhibitor at 1:1000 dilution) twice. The IP samples underwent final washes twice with TE buffer (10 mM Tris-HCl pH 80 and 1mM EDTA, freshly added RNase Inhibitor at 1:1000 dilution). IP samples were eluted from beads with 300 µl elution buffer (50 mM Tris-HCl pH 8, 150mM NaCl, 1 mM EDTA, 1% SDS, 50mM NaHCO3 and freshly added RNase Inhibitor at 1:1000 dilution and 7µl of Proteinase K) at 37 °C for 30 minutes. 500µl Trizol was added to extract RNA and then digested with DNase (Thermo Fisher, NC1424104) at 37 °C for 30 minutes. The Isopropanol precipitated RNA was resuspended in 17.5 µl of RNase/DNase free water at 55 °C for 5 minutes. Libraries were amplified using standard RNA library prep kit by UM AGC. The quantity and quality of the final libraries were assessed using a Bioanalyzer (Agilent Technology Inc). All samples were sequenced by PE150 on the Illumina NovaSeq shared flow cell sequencer. Two biological replicates were conducted for each experiment