GSM9340247: DMSO, RPF, rep3; Homo sapiens; OTHER
Before lysis, cells were incubated with 100 μg/ml cycloheximide (CHX) for 5 min at RT and washed with ice-cold PBS before harvesting by scraping in polysome lysis buffer (20 mM Tris-HCl pH 7.4, 5 mM MgCl2, 150 mM NaCl, 1 mM DTT, 1% Triton-X-100, EDTA-free complete protease inhibitors [Roche]). The lysates were rotated for 10 min at 4°C and cleared from cell debris by centrifugation at 10,000 × g for 10 min at 4°C. For generating ribosome protected fragments, lysates were digested with 240 U RNase I (Ambion) per A260 Unit for 20 min at 4°C. After 17.5–50% [w/v] sucrose-density gradient centrifugation, fractions of ~300 µl were collected during gradient elution and supplemented with 300 µl urea buffer (10 mM Tris, pH 7.5, 350 mM NaCl, 10 mM EDTA, 1% SDS, and 7 M urea) and 300 µl phenol:chloroform:isoamyl alcohol (25:24:1). By phase separation, RNA of the cytoplasmic lysates (inputs; IN) and the monosomal fractions (ribosome protected fragments; RPF) was isolated. The input and RPF samples were depleted of ribosomal RNA (rRNA) using the Human-Mouse-Rat Ribo-Seq riboPOOL kit (siTOOLs Biotech). After random fragmentation of input RNA at 95°C for 12 min in alkaline fragmentation buffer (2 mM EDTA, 88 mM NaHCO3, 12 mM Na2CO3), both input and RPF samples were size-selected (25-35 nt) on 15% polyacrylamide Tris-borate-EDTA-urea gels. Following end-repair using T4 PNK, 1.65 ng per sample were used for library preparation using the NEBNext Multiplex Small RNA Library Prep Set according to the manufacturer's instructions.