title: TTchemseq_DRB_5min_mOHT
organism: Homo sapiens
description: Protocols: TTchemseq: After medium removal and a wash with 1X PBS, 3 ml of TRIzol (ThermoFisher) was added to each 140-cm plate and transferred to 15 ml tubes with the help of a rubber scrapper. Control samples without 4SU or with an overnight incubation with 0.2 mM 4SU were also generated. DRB/TTchem-seq: Cells were harvested with TRIzol as for TTchem-seq.  DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogenInvitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. Total RNA spike-ins were prepared from mid-log phase S. cerevisiae (BY4741 strain) cells grown in YPD + 2% glucose and labeled with 5 mM 4-thiouracil (4TU, Sigma-Aldrich, 440736) for 5 min. AsiSI dependent-DSB were induced by the addition of 300 nM 4-OHT (Sigma, H7904) into the medium for 4 h or 24 h. Etoposide (Sigma, E1383) was used at 50 µM for 30 min for END-seq, 50 µM for 1 h for ChIP. siRNA transfections were performed using 4D-Nucleofector from Lonza either with the SE-small kit for 1 million cells or SE-large kit for 5-20 million of cells, according to the manufacturer's instruction. Cells were collected 48 h after siRNA transfection For TTchem-seq, DSBs were induced by treating DIvA cells with 300 nM OHT for 4 h and nascent RNA was labeled with 1 mM 4-thiouridine (4SU, Glentham Life Sciences, GN6085) for 15 min.  For DRB/TTchem-seq, DSBs were induced by treating DIvA cells with 300 nM OHT for 3 h, followed by a 1 h treatment with 100 μM DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, Sigma-Aldrich, D1916).  DRB inhibition was released by 2 washes with pre-warmed PBS and addition of fresh medium. 4SU labeling was performed directly after DRB release (5- and 10-min time points) or as 10 minutes pulses starting 10, 20 or 30 min after the DRB release (20-, 30- and 40-min time points). Total RNA was isolated using standard chloroform extraction and NaCl/isopropanol precipitation, treated with RQ1 DNAse (Promega, M6101) for 30 min at 37°C and purified by phenol/chloroform extraction and NaCl/isopropanol precipitation. 100 μg of human 4SU-labeled RNA was spiked-in with 1 μg of 4TU-labeled S. cerevisiae RNA. Chemical RNA fragmentation, biotin conjugation and Streptavidin purification were performed according to PMID:31915390 Total RNA was extracted using the PureLink RNA Mini kit (Thermo Fisher Scientific, 12183020, enzymatic protocol, including a 20 min on-column treatment with RQ1-DNase (Promega, M6101)). Sequencing libraries were prepared using the NEBNext Ultra II Directional RNA library prep kit (NEB E7760) omitting the initial fragmentation step