GSM8079965: etohwtr1; Homo sapiens; OTHER
Two days after transfection, unless specified cells were harvested in lysis buffer A (20 mM Tris-Cl [pH 7.5], 100 mM NH4Cl, 10 mM MgCl2, 0.1% Tween20 supplemented with 1X cOmplete™ Mini Protease Inhibitor Cocktail [Roche, 11836170001]). For Figure 4D, cells were trypsinzed and resuspended in PBS. Then the cells were crosslinked with DSP (Thermo Fisher Scientific, PG82081) at 0.5 mM for 30 minutes at RT and quenched by adding Tris-Cl [pH 8] to 50 mM followed by incubation at RT for 15 minutes. After centrifugation at 350 rpm for 5 minutes, cells were lysed in lysis buffer B (50 mM HEPES [pH 7.5], 100 mM NH4Cl, 10 mM MgCl2, 0.1% Tween20 supplemented with 1X cOmplete™ Mini Protease Inhibitor Cocktail [Roche, 11836170001]). 750 µg purified MS2 coat protein was added to lysate containing 1 mg of total RNA, and samples were nutated at 4 ˚C overnight. Add 445 unit of Nuclease P1 (NEB, M0660S) to the lysate and shake in a thermomixer at 37˚C for 30 min. Nuclease-treated lysates were layered on a sucrose cushion (20 mM Tris-Cl [pH 8], 150 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 M sucrose). Ribosomes were pelleted by ultracentrifugation at a TLA100.3 rotor at 100,000 rpm for 1 hour at 4˚C. Resuspend ribosome pellets thoroughly binding buffer (100 mM HEPES [pH 7.5], 200 mM KCl, 5 mM MgCl2, 1 mM DTT, 10 mM BME, 0.02% Tween 20, supplemented with 1X cOmplete™ mini protease inhibitor cocktail). Add 111 µL of pre-equilibrated amylose resin slurry (NEB, E8021) into resuspended ribosomes, and nutate at 4˚C for 90- 120 minutes. Wash resin 10 times with wash buffer (100 mM HEPES [pH 7.5], 400 mM KCl, 5 mM MgCl2, 1 mM DTT, 10 mM BME, 0.02% Tween 20, 5% ethanol). Elute tMS2-tagged ribosomes in elution buffer (100 mM HEPES pH 7.5, 200 mM KCl, 5 mM MgCl2, 1 mM DTT, 10 mM BME, 0.02% Tween20, 10 mM maltose, supplemented with 1X cOmplete™ Mini Protease Inhibitor Cocktail) with nutation at 4˚C for 30 minutes. Tenth of eluate was then extracted with hot acidic phenol/chloroform and precipitated for primer extension analysis. Footprints were extracted and size-selected on 15% TBE-urea gels between 40-80 nt. The rest of library construction procedures was as described previously3. Libraries were sequenced on a NextSeq 1000 machine. Libraries were generated by using error-prone reverse transcription to generate cDNA.  Then libraries underwent gene specific  amplification to adding Illumina adaptors, and a second amplification to add Illumina barcodes.