GSM8712382: hcc1143_ANS_10uM_rep3; Homo sapiens; OTHER
Cells were washed with ice-cold phosphate-buffered saline and lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1mM DTT, 1% v/v Trizon X-100, 25 U/mL Turbo DNase I). Lysates were incubated on ice for 30 minutes to allow for DNase digestion and clarified by centrifugation at 20,000 × g for 30 minutes at 4 °C. Lysates were digested with 3.33 U/µg RNase I (Ambion) for 45 minutes at room temperature with gentle agitation. Resulting monosomes were isolated by sucrose cushion, using 450 µL 1M sucrose in polysome buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1mM DTT) for 400 µL of digested lysate material. Sucrose cushions were performed with MLA-130 rotor in an Optima Max-XP ultracentrifuge (Beckman Coulter) at 100,000 × g for 90 minutes at 4 °C. Pellet were resuspended in resuspension buffer (150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 20 mM pH 7.5 Tris-HCl) and incubated at room temperature for at least 10 minutes. RNA was extracted using Direct-zol RNA miniprep kit (Zymo), according to manufacturer's instructions. Ribosome footprint RNA size selection was performed as previously described (McGlincy & Ingolia 2017, Methods) with minor modifications, including the use of 10% TBE-urea gel (Biorad) and custom sizing markers (oIB188-190, 17nt-34nt). Following overnight gel extraction and ethanol precipitation, RNA was dephosphorylated with T4 PNK (NEB) at 37C for 1 hour. Ribosome footprint cDNA libraries were subsequently constructed with Ordered Two-Template Relay kits (Karnateq) according to manufacturer's recommendations (Ferguson et al. (2023), Nat. Methods). cDNA libraries were indexed and amplified with NEBNext Indexing Primers (NEB).