a, The co-IP of endogenous SETX, BRCA1, Dicer or AGO1 using HeLa nuclear extracts shows that RAD51, an established BRCA1-binding partner involved in homologous recombination, did not co-immunoprecipitate with SETX and co-immunoprecipitated very weakly, if at all, with Dicer and AGO1. Representative blot from n = 3 experiments. b, Endogenous BRCA1 co-IP of HeLa nuclear extracts using two BRCA1 antibodies, each of which recognizes a different epitope (Ab#1, N terminus; Ab#2, C terminus). Representative blot from n = 3 experiments. c, BRCA1 co-IP performed using nuclear extracts from BRCA1 siRNA (siBRCA1)-transfected HeLa cells. Representative blot from n = 3 experiments. d, e, co-IP of endogenous BRCA1 and RNAi factors that are present in subcellular HeLa fractions (d) and confirmed in HMECs and human fibroblasts (BJ-hTert) (e). Representative blot from n = 4 experiments. Chrom, chromatin; Cyt, cytoplasm; NS, nuclear soluble. f, BRCA1 was co-immunoprecipitated, using three monospecific antibodies, in HeLa nuclear extracts from cells overexpressing (+) or not overexpressing (–) RNase H1. Immunoblots showed using antibody 2 (Ab#2) that a BRCA1–SETX–RNAi complex was R-loop sensitive. Representative blot from n = 2 experiments. g, Dicer, AGO1 and AGO2 ChIPs were performed in BRCA1-depleted (BRCA1-KD) HeLa cells. Recruitment of Dicer, AGO1 and AGO2 to ACTB pause sites was BRCA1-dependent (n = 2–5 biological replicates). h, Representative immunoblots demonstrating siRNA-mediated silencing efficiency in HeLa cells and HMECs. Representative blot from n = 10 experiments. i, Representative DNA damage analysis using separate siRNAs for each relevant target. j, DNA damage analyses at ENSA (R-loop-positive) and AKIRIN1 CoTC (cotranscriptional cleavage, R-loop-negative) transcription termination sites (n = 3–7 biological replicates). k, The BELD strategy used to identify DNA breaks at the ACTB pause site. l, m, BELD quantification of breaks at the ACTB pause site. l, BELD performed at the ACTB pause site in BRCA1-depleted cells with or without prior in vitro nick and gap repair (±NGR) of breaks performed before terminal deoxynucleotidyl transferase labelling. m, BELD performed on the reverse strand of the ACTB pause site. A representative example of each BELD analysis is shown, and the histograms depict the mean ± s.d. values of qPCR replicates. All ChIP data are represented as an mean ± s.e.m. fold change of the relevant knockdown condition compared to the mock condition. g, i, j, Data were analysed using multiple t-test (g), two-way ANOVA with post hoc Tukey HSD and unpaired t-test (i), and compared to results obtained with the undamaged locus or with relevant control cells (j).