Endogenous RNA:DNA hybrids were immunoprecipitated using the S9.6 antibody. Briefly, HeLa cells were crosslinked with 1% paraformaldehyde (PFA) for 20 min at room temperature. PFA was quenched by incubation with 125 mM glycine for 5 min at room temperature. Cells were harvested and lysed in TENTG-150 on ice for 30 min. Lysates were centrifuged at 13,000 rpm at 4°C for 30 min. The soluble fraction was precleared for 20 min at 4°C with 15 μl of agarose protein G beads, followed by incubation overnight at 4°C with 30 μl of agarose protein G beads coupled to 10 μg/ml of either irrelevant mouse immunoglobulin G (IgG) or S9.6 antibody. Beads were washed five times in TENTG-150 buffer, and immunoprecipitated material was eluted in Laemmli buffer.

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