For stable integration of H2AFX gene constructs into HeLa-FlpIn cell line, the pcDNA5-FRT-TO-eGFP-Linker plasmid, kindly provided by the Castello laboratory (Oxford University), was employed as backbone. The region containing the CMV promoter up to after the bGH (bovine growth hormone) poly(A) site was excised using MluI and SphI restriction enzymes (New England Biolabs (NEB, #R0198S and #R0182S). The linearised plasmid was gel-purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research, #D4007) and dephosphorylated using the FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, #10819230). For the insert, the H2A.X gene was amplified from HeLa genomic DNA using 5’-CTATCGacgcgtTTCCCAGACGCTCTCTAGGT-3’ as forward primer, binding 335 base pairs (bp) upstream of the H2AFX 5’UTR, thus including the H2AFX promoter, and con- taining the MluI restriction site. As reverse primer, 5’-TTACATgcatgcGTTCTCCTGGTAC GTCCTTTCT-3’ was employed, binding 1807 bp downstream of the H2AFX 5’UTR start, including the entire H2AFX gene with its own poly(A) site and containing the SphI restriction site. The PCR was performed with Q5 polymerase (NEB, #M0491L), HeLa genomic DNA as template. The PCR product was gel-purified, digested with MluI and SphI, gel purified once more and ligated into the linearised and dephosphorylated plasmid. Ligation was performed using T4 DNA ligase (NEB, #M0202S). The ligated product was transformed into E. coli XL1-Blue competent cells. Positive clones were selected by colony PCR and Sanger Sequencing using primers listed in Supplementary Table 2. The resulting plasmid called pcDNA5-FRT-H2AFX wt was employed as a template to make H2AFX mutant constructs by site-directed mutagenesis.

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