title: GSM5100356: BrUseqRep3_WT8h_SSPE_TagDir; Homo sapiens; OTHER
library_construction_protocol: ATAC-seq: For each experimental condition 50.000 cells were harvested and washed with cold PBS containing protease inhibitors (Roche). The cells were resuspended in 50 µl Transposase mixture, containing 1x Tagment DNA buffer Illumina Kit, #20034197), 0.5 µl Tagment DNA enzyme (Illumina Kit, #20034197) and 0,2 µg/µl Digitonin (Promega, #G9441), and incubated at 37 °C for 30 min. Fragmented DNA was purified using a Qiagen MinElute kit (#28204).  BrU-seq: For BrU(DRB)-seq, cells were pre-treated with 100 µM DRB for 1-3hrs (as indicated per sample) after which cells were washed with PBS and immdeidately, or 30 min later, incubated with 2 mM bromouridine (Bru) at 37°C for 15 or 30 min. For regular BrU-seq, cells were either mock-treated or irradiated with UV-C light (9 J/m2). Cells were then incubated in conditioned media for different periods of time 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: Cells were mock treated or UV-irradiated with 9 J/m2 and incubated in conditioned media for different periods of time (1, 4, 8, 16 h). Cells were 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. Chromatin was sonicated to approximately 1 nucleosome using a Bioruptor waterbath sonicator (Diagenode). Chromatin of ~5x106 cells was incubated with 3 µg antibody (Antibodies are listed in Table S3) overnight at 4°C, followed by a 1.5 h 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 h at 65°C in the presence of proteinase K. DNA was purified using a Qiagen MinElute kit.  RNA-seq: Cells were UV-irradiated (254 nm, 9 J/m2) or mock-treated and harvested 24 h later. Total RNA was isolated using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Up to 5 × 106 cells per sample were lysed in RLT buffer. For quantification purposes, 24 ng of the eight ArrayControl™ RNA Spikes (Thermo Fisher Scientific) were added to each sample (3 ng/spike). ATAC-seq: Libraries were prepared by PCR using NEBNext High-Fidelity 2x PCR Master Mix (New England Labs, #M0541) and Nextera XT index Kit v2 (Illumina). Samples were sequenced on an Illumina HiSeq X system (Macrogen), resulting in 150 bp paired-end reads. 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 Advanced Genomics Core. Indicated is whether single-end or paired-end sequencing data was used for downstream analyses ChIP-seq: For RNAPII-Ser2 ChIP-seq sample libraries were prepared from 1 ng ChIPed DNA using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S) and index primer sets (NEB, E7335S). Strand-biased library amplification was performed using high-fidelity KAPA HiFi Enzyme (Roche, KK2102). The prepared libraries were sequenced on an Illumina HiSeq 2500 system (Illumina), resulting in 150 bp paired-end reads. For the remaining ChIP-seq samples, libraries were prepared using kapa hyper prep kit (Roche) and A-T mediated ligation of full Y-shaped IDT adapters. Samples were sequenced on an Illumina HiSeq X system (Macrogen), resulting in 150 bp paired-end reads. RNA-seq: Samples were purified using the KAPA mRNA Hyperprep kit (Roche) and prepared for sequencing using the TruSeq RNA Library Prep Kit v2 (Illumina) according to the manufacturer's instructions, and sequenced in a single lane on an Illumina HiSeq4000.