title: Riboseq of HepG2: rep3
design: An equivalent to 8 OD260 of lysate was treated with 66 U RNase I (Invitrogen) for 45 min at 22C in a thermomixer with mixing at 1000 rpm. Then, 200 U SuperaseIN RNase inhibitor (20 U/l, Invitrogen) was added to each sample. Digested lysates were loaded onto 10-50% home-made sucrose density gradients in open-top polyclear centrifuge tubes (Seton Scientific). Tubes were centrifuged at 35000 rpm for 3 hours at 4C (SW-41 Ti rotor, Beckmann Coulter ultracentrifuge. Samples were fractionated using the Piston Gradient Fractionator (Biocomp Instruments) at 0.75 ml/min by monitoring A260 values. Thirty fractions of 0.37 ml were collected in 1.5 ml Eppendorf tubes, flash frozen, and stored at -80C. The fractions (typically 3 or 4) corresponding to the digested monosome peak were pooled. RNA was extracted using the hot acid phenol/chloroform method. The ribosome-protected RNA fragments (28-32 nt) were selected by electrophoresis on 15% polyacrylamide urea TBE gels and visualized with SYBR Gold Nucleic Acid Gel Stain (ThermoFisher Scientific). Size selected RNA was dephosphorylated by T4 PNK (NEB) for 1 h at 37C. RNA was purified using the acid phenol/chloroform method. Depletion of rRNA was performed using the riboPOOL kit (siTOOLs biotech) from 433 ng of RNA according to the manufacturers instructions. Libraries were prepared using the SMARTer smRNA-Seq Kit for Illumina (Takara) following the manufacturers instructions from 15 ng of RNA. Libraries were purified by electrophoresis on 8% polyacrylamide gels and sequenced on the Illumina NextSeq 500 sequencer in the Genomics Facility Basel (Department of Biosystems Science and Engineering (D-BSSE), ETH Zrich).