GSM8325894: USP42_KO_RNA_rep1; Homo sapiens; RNA-Seq
Three million cells for the PARK7, USP42, and VIM knockout cell lines, along with a AAVS1 (safe harbor control) knockout line, were plated in three 10 cm2 dishes. 27 h later cells at ~60% confluency were treated with 100 µg/mL cycloheximide (CHX) for 10 min at 37 ˚C, then collected in ice cold PBS with 100 µg/mL CHX. Cells were spun at 100 x g for 7 min at 4 ˚C, then flash frozen in liquid nitrogen and stored at -80 ˚C. Cell pellets were lysed with 400 µL lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton-X, 100 µg/mL CHX, 1x protease inhibitor EDTA free) for 10 min on ice. Lysates were clarified by centrifugation at 1,300 rpm for 10 min at 4 ˚C. 40 µL lysate was saved for total RNA extraction, and the rest of the lysate was digested with 7 µL RNaseI for 1 h at 4 ˚C. Digestion was stopped by adding ribonucleoside vanadyl complex to a final concentration of 20 mM. Digested lysates were then loaded onto 10 mL sucrose cushion (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 M sucrose) and centrifuged at 38,000 rpm for 2.5 h at 4 ˚C using a SW41-Ti rotor. The pellet and the total RNA aliquot were both solubilized with 1 mL Trizol, and RNA was purified with the Zymo Direct-zol RNA Miniprep Kit, including DNaseI digestion. RNA-seq: Quality of total RNA was confirmed with Bioanalyzer RNA Pico. All RIN scores were >= 9.8. Libraries were prepared from 1 µg total RNA using the NEBNext Ultra II RNA Library Prep Kit for Illumina according to manufacturer's protocol and using 8 cycles for PCR. Ribosome profiling: Ribosome protected fragments (RPFs) were size-selected on a 15% TBE urea gel by electrophoresing at 150 V for 1.5 h. RPFs between 28-32 nt were sliced, using 28 nt and 35 nt markers as a guide. Slices were frozen at -20 ˚C for 1 h, crushed with pestles, and the RPFs were eluted in gel extraction buffer (300 mM sodium acetate pH 5.5, 5 mM MgCl2) by rotating overnight at room temperature. Eluates were passed through Costar Spin-X filter tubes at 12,000 x g for 1 min 30 s. Then 1 µL 1 M MgCl2, 2.5 µL GlycoBlue, and 1 mL ethanol were added and the RPFs precipitated for two days at -20 ˚C. Pellets were dried and resuspended in 16 µL water. Libraries were generated from 8 µL RPF eluate using the Diagenode D-Plex Small RNA Kit with minor modifications: in the 3' dephosphorylation step 0.5 µL T4 PNK was supplemented and incubated for 25 min. The RTPM reverse transcription primer was used and 8 cycles were performed for PCR. Libraries were quantified by Bioanalyzer High Sensitivity DNA Kit, pooled equimolar according to the quantity of the peak for libraries with full-length inserts (~204 nt), and cleaned up with 1.8X AMPure XP beads. Adapter dimers and empty libraries were removed by size-selection on a 12% TBE PAGE gel, followed by extraction with the crush and soak method, and final libraries were resuspended in 20 µL water.