DNA- and RNA-ChIP

ChIP assays were performed as described^31,48, with some modifications. Briefly, mock-, GO-, Bleo-treated or IR-exposed cells were crosslinked in 1% formaldehyde for 10 min at room temperature and sonicated to an average DNA size of ∼300 bp in 50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 1% SDS with 1X protease inhibitor cocktail using a Qsonica sonicator. The supernatants were diluted with 15 mM Tris-HCl, pH 8.0, 1.0 mM EDTA, 150 mM NaCl, 1% Triton-X-100, 0.01% SDS and protease inhibitors and incubated with appropriate Abs overnight at 4 °C. ICs were captured by Magna ChIP Protein-A magnetic beads (Millipore) that were then washed sequentially in buffer I (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100 and 0.1% SDS); buffer II (same as buffer I except containing 500 mM NaCl); buffer III (1% NP-40, 1% sodium deoxycholate, 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA), and finally with 1X Tris-EDTA (TE, pH 8.0) buffer. The ICs were extracted from the beads with elution buffer (1.0% SDS and 100 mM NaHCO₃), de-crosslinked for 4 h at 65 °C, and DNA isolated by phenol–chloroform extraction and ethanol precipitation using GlycoBlue (Life Technologies) as carrier. For RNA-ChIP, a similar protocol was followed with minor changes. Briefly, after crosslinking NEs were prepared before sonication. The cells were incubated in buffer A (5 mM HEPES, 85 mM KCl, 0.5% NP-40 and 1X Protease inhibitor) for 10 min at 4 °C, then washed once with buffer B (buffer A minus NP-40) at 2,500g for 5 min and nuclear pellet was resuspended in sonication buffer. 50 U ml⁻¹ of RNase inhibitor (Roche) was added to buffers A and B, sonication and IP buffers, and 40 U ml⁻¹ to each wash buffer. ICs were captured using Protein A/G PLUS agarose beads, and the de-cross-linking time was reduced to 2 h. Isolation was carried out in acidic phenol–chloroform followed by ethanol precipitation with GlycoBlue as a carrier. Genomic DNA was removed and reverse transcription performed using a PrimeScript RT Kit with gDNA Eraser (TaKaRa). ChIP and RNA-ChIP samples were analysed by PCR/qPCR using specific primers (Supplementary Table 1). qPCR data are represented as per centage input after normalization to IgG (or as fold increase in normalized per cent input, in the case of heat-shock-related experiments). For RNA-ChIP no amplification was detected with IgG, so the per cent input for IgG was taken as zero, and the data were represented as per centage input for the rest of the samples. Antibodies used for DNA/RNA-ChIP were the same as listed for co-IP analysis except for the Histone H2A.XS 139 ph (ser phos 139 residue; GTX628789, GeneTex) used for γH2AX ChIP.

Validation and analysis of Lig IV and Lig IIIα association in transcribing versus non-transcribing regions were performed by genomic induction of DNA DSB with the CRISPR/Cas9 system at sites A and B, which are transcribing and non-transcribing regions on chromosome number 1 of human U2OS cells. After induction of DSBs at these sites, ChIP assays were performed as described³⁵. Primers for qPCR are also located 0.2 and 1.5 kb from the break, as a negative control locus. Values were represented as relative levels of association of Lig IV/Lig IIIα.