GSM8331547: HepG2 siControl Input rep3; Homo sapiens; OTHER
The cell was added with 0.4 mL lysis buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT, 0.1 mg/mL cycloheximide, 0.1 U/µL Invitrogen Turbo DNase). The cell was scraped and collected into 1.5 mL tubes, and then incubated on ice with periodic inversions for 10 min. The lysate was clarified by centrifugation for 10 minutes at 20,000 xg at 4 °C. 0.3 mL supernatant was transferred to a fresh tube. 300 U of RNAse I was added and the lysate was incubated at room temperature for 45 min. Afterwards, 15 μL Invitrogen SUPERase·In™ RNase Inhibitor (20 U/μL) to stop this reaction. The 0.3 mL of lysate was transferred to a prewashed MicroSpin S-400 column (Sigma: Microspin™ S-400 HR). The column was centrifuged at 600xg for 2 min to collect flow-through. Zymo RNA Clean & Concentrator Kit was used to purify RNA. The 28-30 nt RPFs (Ribosome-protected fragments) were excised from 10% TBU gel and recovered by ZR small-RNA™ PAGE Recovery Kit. The RPFs were end-repaired to have a 5'-phosphate and 3'-OH by T4 PNK treatment. The rRNA depletion was performed using customed antisense biotin-labeled DNA oligo. The library was prepared by NEB small RNA library preparation kit.