Figure 3

Aberrant accumulation of DNA:RNA hybrids at pericentromeric repeats in ICF mutant cells. (a) Validation of DRIP qPCR procedure. The efficiency of immunoprecipitation was validated with two previously known R-loop positive loci, TFPT and CALM3, and two negative loci, EGR1 and SNRPN³⁰, in RNase H untreated WT cells. The positive loci were successfully immunoprecipitated, whereas the Ct values of the negative loci were below the detection threshold. Amount of the immunoprecipitants was shown as percent input. ND, not detected. (b) Results of DRIP qPCR of satellite-2 and α-satellite in WT and CDCA7 KO cells. DNA was extracted from these cells and treated with or without E. coli RNase H in vitro. Amount of the immunoprecipitants was shown as percent input. Experiments were conducted in biological duplicate and technical triplicate. (c) Validation of R-ChIP qPCR procedure. The efficiency of immunoprecipitation was validated with the two R-loop positive loci and two negative loci in WT cells. The positive loci were successfully immunoprecipitated, whereas the Ct values of the negative loci were below the detection threshold. Amount of the immunoprecipitants was shown as percent input. ND, not detected. (d) The V5–RNASEH1_D210N expressing plasmid vector was transfected with WT, DNMT3B KO, ZBTB24 KO, CDCA7 KO, and HELLS KO HEK293 cells, and R-ChIP qPCR was conducted using anti-V5 antibody 48 h after transfection. Relative amounts of immunoprecipitants with anti-V5 antibody were detected using primers for amplifying satellite-2 and α-satellite. Since transfection efficiency of the plasmid vector could be different between the cells, the ampicillin-resistant gene (Amp^R) in the vector in input was used for normalization. The amount of immunoprecipitants of WT was considered to be 1.0. Experiments were conducted in biological triplicate and technical triplicate. Data are mean ± s.e. P values were obtained using the Mann–Whitney two-tailed U test.