title: SUM159_LeptomycinB_Polysomal_Rep1
design: CELL CULTURE:  SUM159-CRISPRi cells (PB TRE dCAS9-KRAB) - from here on named SUM159 - were cultured in HAMs-F12 (Thermofisher 11765054) with 5% TET free serum, insulin (5 g/ml), hydrocortisone (1 g/ml), 10 mM HEPES and 100 ug/mL Hygromycin B. Cells were grown at 37C and 10% carbon dioxide. METABOLIC LABELING: SUM159 cells were treated with 500uM of 4-Thiouridine (Santa Cruz CAS 13957-31-8) for 20 minutes. TREATMENTS: 100nM of Pladienolide B (Santa Cruz CAS 445493-23-2) for 4h OR 20ng/ml of Leptomycin B (Santa Cruz CAS 87081-35-4) for 18h OR 1ug/ml of Harringtonine (Abcam 26833-85-2) for 1h. POLYSOMAL FRACTIONATION: Cells were treated with Cycloexamide 100g/ml (Merk C4859) for 10 min before lysis then washed twice with ice-cold PBS + cyclohexamide 10g/ml. Cells were lysed in 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.1% NP-40, 100g/ml cycloheximide, 1mM DTT, 0.2mg/ml heparin and let 10 minutes on ice. After centrifugation at 12,000 r.p.m. for 10 min at 4 C, cytoplasmic extracts were loaded on a 1550% sucrose gradient and centrifuged at 4 C in a SW41Ti Beckman rotor for 3 h 30 min at 39,000 r.p.m. Absorbance at 254 nm was recorded by BioLogic LP software (BioRad). RNA EXTRACTION: RNA from polysome fraction was extracted using Qiazol and mRNA purification was performed with up to 200ug of Total RNA using MACS mRNA Isolation Kit (Miltenyi Biotec 130-075-201). LIBRARY PREPARATION: Nanopore Direct RNA sequencing libraries were prepared using up to 200ng of mRNA as an input.