title: GSM4190625: data_TIR1_6J_2_Total; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-seq: Cells were plated and grown to ~90% confluency and crosslinked with 0.5 mg/mL disuccinimidyl glutarate (DSG; Thermo Fisher) in PBS for 45 min at room temperature. Cells were washed with PBS and crosslinked with 1 % PFA for 20 min at room temperature. Fixation was stopped by adding 1.25 M Glycin in PBS to a final concentration of 0.1 M for 3 minutes at room temperature. Cells were washed with cold PBS and lysed and collected in a buffer containing 0.25% Triton X-100, 10 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 20 mM Hepes (pH 7.6). Chromatin was pelleted in 5 min at 400 g and incubated in a buffer containing 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 50 mM Hepes (pH 7.6) for 10 minutes at 4°C. Chromatin was again pelleted for 5 min at 400 g and resuspended in ChIP-buffer (0.15 % SDS, 1 % Triton X-100, 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 20 mM Hepes (pH 7.6)) to a final concentration of 15x106 cells/ml. BrU-seq: U2OS osTIR1 or PAF1-AID cells were induced with doxycycline for 24 hrs, and subsequently with auxin for 5 hrs. After this treatment, cells were either mock-treated or irradiated with UV-C light (7 J/m2). Cells were then incubated in conditioned media for different periods of time (0, 3, 8, 24h) before being incubated with 2 mM bromouridine (Bru) at 37°C for a 30 min. The cells were then lysed in TRIzol reagent (Invitrogen) and Bru-containing RNA isolated as previously described (Andrade-Lima et al., 2015). ChIP-seq: Chromatin was sonicated to approximately 1 nucleosome using a Bioruptor waterbath sonicator (Diagenode). Chromatin of ~5x106 cells was incubated with 3ug antibody (RNAPII, rabbit polyclonal, Bethyl laboratories, A304-405A; PAF1, rabbit polyclonal, Bethyl laboratories, A300-172A) over night at 4°C, followed by a 1.5 hrs protein-chromatin pull-down with a 1:1 mix of protein A and protein G Dynabeads (Thermo Fisher; 10001D and 10003D). ChIP samples were washed extensively, followed by decrosslinking for 4 hrs at 65°C in the presence of proteinase K. DNA was purified using a Qiagen MinElute kit. Sample libraries were prepared using Hifi Kapa sample prep kit and A-T mediated ligation of Nextflex adapters. Samples were sequenced using an Illumina NextSeq500, using paired-end sequencing with 42 bp from each end. BrU-seq: cDNA libraries were made from the Bru-labeled RNA using the Illumina TruSeq library kit and paired-end 151 bp sequenced using the Illumina NovaSeq platform at the University of Michigan DNA Sequencing Core. Only single-end sequencing data was used for downstream analyses
attributes.tag: GEO Accession
attributes.value: GSM4190625