title: SUM159_Harringtonine_Cytoplasmic_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. CELLULAR FRACTIONATION: Cells were tripsinized and resuspended, counted, centrifugated at 1200rpm for 5 min, washed with ice cold PBS with RNAse inhibitor and the pellet was resuspended in Cytoplasmic lysis buffer for the subsequent fractionation, following the protocol described in https://doi.org/10.1016/j.cell.2012.05.043. RNA EXTRACTION: RNA in the three subcellular fractions was purified using Qiazol (Qiagen 79306). Total RNA was quantified 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.