library_construction_protocol: U2OS and DIvA U2OS cells were plated in 15 cm dishes at densities of 4.5 million cells in complete DMEM, supplemented with 5 μM 5-azacytidine and incubated for 12 hours. The following day, DNA double-strand breaks (DSBs) were induced using AsiSI by adding 4-hydroxytamoxifen to complete DMEM achieving a final concentration of 500 nM. The cells were incubated for 4 hours at 37°C. Ten minutes before the end of DSBs induction, DSP (dithiobis[succinimidyl propionate]) was freshly dissolved in DMSO at concentration of 100 mM and then diluting dropwise into 150 ml of pre-warmed PBS containing Ca²⁺/Mg²⁺ while stirring at 37°C, to a final concentration of 1 mM. After DSB induction, cells were washed once with PBS containing Ca²⁺/Mg²⁺, then incubated with DSP solution for 30 minutes at 37°C. Subsequently, formaldehyde was added directly to the media (final concentration 1%) and incubated for 15 minutes at 37°C. Crosslinking was quenched with Glycine (final concentration 125mM) for 15 minutes at room temperature. Cells were washed twice with ice-cold PBS without Ca²⁺/Mg²⁺, collected by scraping using ice-cold PBS containing 0.5% BSA and pelleted by centrifugation at 500 g for 5 minutes at 4°C. Then, pellets were gently resuspended in ice-cold Cell Lysis Buffer (CLB) (85 mM KCl, 5 mM PIPES, 0.5% NP-40, 1× Halt protease inhibitor cocktail (PI), and 1 mM PMSF in DEPC-treated water), prepared immediately prior to use. Cells were incubated on ice for 10 minutes, pelleted by centrifugation at 500 g for 5 minutes at 4°C, and then resuspended in Nuclear Lysis Buffer (NLB) (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1× Halt PI, and 1 mM PMSF, in DEPC-treated water). Nuclei were incubated at room temperature for 10 minutes prior to sonication. Chromatin was sheared using a Diagenode PICO Bioruptor at ultra-high power, with 30-second ON/OFF cycles for 30 minutes. The sonicated lysate was centrifuged on a table top centrifuge at 17,200 RCF for 10 minutes at 4°C to pellet insoluble material. The soluble chromatin was pre-cleared by incubating with 1:1 mixture of Protein A and Protein G magnetic beads in ChIP IP buffer (16.7 mM Tris-HCl pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 0.01% SDS, 1.1% Triton X-100, 1× Halt PI, and 1 mM PMSF in DEPC-treated water) and incubated for 1 hour at 4°C with rotation. Each sample was then split into input controls, antibody samples, no-antibody control. The antibody samples received 5 μg of NSUN2 mouse monoclonal antibody (1 μg/μl), then were incubated overnight at 4°C with rotation at 16 rpm. On the following day, antibody-chromatin complexes were captured by conjugation to freshly washed Protein A/G magnetic beads. Beads were washed twice in ChIP IP buffer containing protease inhibitors and subsequently added to each sample and incubated for 2 hours at 4°C with rotation at 16 rpm. Beads were then subjected to a sequential wash with ChIP wash buffers A-D, each prepared fresh in DEPC-treated water and pre-chilled. Buffer A (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, and 1% Triton X-1000. Buffer B (500 mM NaCl 20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, and 1% Triton X-1000). Buffer C (10 mM Tris-HCl pH 8.0, 2 mM EDTA, 250 mM LiCl, 1% sodium deoxycholate, and 1% NP-400. Buffer D (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). Each wash was performed for 5 minutes at 4°C with rotation, followed by 3 minutes on a magnetic rack. Buffers A–C were used once, and buffer D was applied twice, transferring beads to fresh tubes during the final wash. Elution was carried out by incubating beads twice with 250 μl of Elution Buffer (100 mM NaHCO₃, 1% SDS in DEPC-treated water) at 65°C for 15 minutes on thermomixer with shaking at 1200 rpm, pooling eluates in fresh tubes. To perform digestion and de-crosslinking, Digestion Buffer (10×) containing 400 mM Tris-HCl pH 6.5 and 100 mM EDTA was added. RNase A (50 μg final concentration) was added to all samples and incubated at 60°C for 3 hours on thermomixer at 400 rpm. Afterward, NaCl and DTT were added to final concentrations of 300 mM and 5 mM, respectively. Proteinase K (50 μg final concentration) was then added and samples were incubated overnight at 65°C on thermomixer with shaking at 400 rpm. The following day, DNA was purified via phenol/chloroform extraction. DNA precipitation was performed adding Isopropanol (0.8 volumes), NaCl (300mM final concentration), MgCl₂ (10mM final concentration), and GlycoBlue (30μg final concentration), and samples were incubated for 2 hours at -20°C. DNA was pelleted by centrifugation on a table top centrifuge at 17,200 RFC for 30 minutes at 4°C. Pellets were washed twice with 100% ethanol and 70% ethanol. Then, pellets were air-dried for 2 hours and resuspended in 50 μl of TE buffer with low EDTA (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA in DEPC-treated water). DNA was quantified using the Qubit dsDNA High Sensitivity assay. ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.