ChIP–seq

TIG3 cells for the indicated conditions were cross-linked for 10 min in 1% formaldehyde and chromatin was fragmented by sonication using Bioruptor Sonicator (Diagenode). Chromatin immunoprecipitation was performed as previously described⁹⁹ with antibodies against H3K9me3 (5 µg, Abcam ab8898) and H3 (2 µg, Abcam ab10799). The immunoprecipitated DNA was quantified by Qubit fluorometer (Life Technologies). DNA library for Illumina sequencing was prepared from 10 ng DNA, using NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs) and following the manufacturer’s instructions. Equimolar amounts of samples, with compatible indexes, were pooled for multiplex sequencing. For all samples, single-end sequences were generated on the Illumina HiSeq2000 platform at the Danish National High-throughput DNA Sequencing Centre.

ChIP–seq data are available at the Gene Expression Omnibus (PRJNA897702). Raw reads were aligned to the human genome (hg19 assembly excluding non-canonical chromosomes that is random, unknown and haplotype variant chromosomes) using Bowtie version 0.12.7 with default parameters except ‘-S -m 1’, which excludes reads mapping to multiple chromosomal positions. Peak detection was performed with MACS2 version 2.0.9 (20111102) using default settings except for parameters ‘–broad–nomodel–shiftsize=110’. The shift size of 110 bp was calculated as the median over all Phantom Peak¹⁰⁰ shift estimates for our H3K9me3 samples. When running differential peak detection between two H3K9me3 samples in MACS2 the additional parameter ‘–shift-control’ was specified. Bigwig files were generated using the UCSC Kent utilities¹⁰¹. We allowed only one read per chromosomal position thus eliminating potential spurious spikes, and each remaining read was extended from its 5′-end to a total length of 250 bases, before converting to bedGraph format, scaling to mapped reads per million and final conversion to bigwig format. Individual BigWig files were uploaded to the UCSC browser for visualization^101,102. To generate chromosome-wide landscapes of H3K9me3 and H3 we used the mean as the combining function and a smoothing window of 4 pixels. Overlay plots were generated by creation of a track hub at UCSC browser¹⁰³, where individual BigWig tracks were combined into a multiWig display with two coloured transparent graphs overlaid in the same vertical space. We used the integrative analysis tools from the Cistrome platform¹⁰⁴ to calculate Pearson correlation coefficients for multiple signal profiles on a whole-genome scale using non-overlapping windows of 250 bp. The association of H3K9me3 peaks with annotated genomic features was calculated using the Cis-regulatory Element Annotation System (CEAS) package¹⁰⁵. Hilbert curve visualization of ChIP–seq data was generated using the HilbertVis application³⁴. Hilbert plots allow the visualization of linear sequence data in two-dimensional space. Each coloured spot in the figure correspond to a peak where the area of the spot is proportional to the width of the peak and the intensity of the spot corresponds to the height of the peak.

Total RNA was extracted using the ReliaPrep RNA Miniprep Systems (Promega) according to the manufacturer’s instructions. Five-hundred nanograms of total RNA was used for mRNA sequencing preparation using the Quantseq 3′mRNA kit following the manufacturer’s protocol. NGS (next-generation sequencing) short reads were aligned to the GRCh38 human genome using the Star aligner. The log₂ fold change in gene expression relative to wild type for each sample was computed from read counts using DEGSeq, and box plots were produced using the R packages.

Total histones from TIG3 cells were isolated by acid extraction. Digestion and mass spectrometry analyses were performed as described in ref. ³¹. The relative quantification for a given peptide was obtained by dividing its quantification by the sum of all quantifications of all peptides sharing the same amino acid sequence. The mass spectrometry raw data are available upon request.

For iPOND experiments, heavy lysine- and arginine-labelled mESCs were pre-treated with UNC0642 at a concentration of 1 µM 2 h before the beginning of the experiment. Light lysine- and arginine-labelled mESCs were pre-treated with same amount of dimethyl sulfoxide (DMSO) at the same time. Both light- and heavy-labelled mESCs were then incubated with 10 µM EdU for 10 min, with and without treatment of 4 mM HU (Sigma-Aldrich) for 3 h to stall the DNA replication forks. After labelling and treatment cells were cross-linked with 1% formaldehyde for 10 min at room temperature, quenched with 0.125 M glycine, washed with PBS and collected using cell scrapper. Samples were then treated with Click-it reaction containing 25 µM biotin-azide, 10 mM (+) sodium l-ascorbate and 2 mM CuSO₄ and rotated at 4 °C for 1 h. Samples were then centrifuged to pellet down the cells; supernatant was removed and replaced with 1 ml Buffer-1 (B1) containing 25 mM NaCl, 2 mM EDTA, 50 mM Tris–HCl, pH 8.0, 1% IGEPAL and protease inhibitor and rotated again at 4 °C for 30 min This step was repeated twice. Samples were centrifuged to pellet down the cells; supernatant was removed and replaced with 500 μl of B1 and sonicated using a Bioruptor Sonicator (Diagenode) using cycles of 20 s on, 90 s off 30 times at high amplitude. Samples were centrifuged, and supernatant was transferred to fresh tubes and incubated for 1 h with 200 μl of Dynabeads MyOne C1 (Sigma-Aldrich) for the streptavidin biotin capture step. Proteins were eluted, and mass spectrometry was performed. At least two peptides were required for protein identification. Quantitation is reported as the log₂ of the normalized heavy/light ratios with respect to mcm6. SILAC data were analysed using Proteome Discoverer (ThermoFisher).

Cells were seeded in triplicate in 10 cm culturing dish and treated with different concentrations of olaparib throughout the whole experimental process, or different concentrations of cisplatin, for 4 h before being washed off and replaced with new medium.

After 1 week, colonies were fixed and stained in a mixture of 43% water, 50% methanol, 7% acetic acid and 0.1% Brillant Blue R (Sigma-Aldrich) and subsequently counted with Gelcount (Oxford Optronix). The survival was plotted as the mean percentage of colonies detected following the treatment normalized to the mean number of colonies from the untreated samples.