Quantified strand-specific DNA:RNA immunoprecipitation sequencing on T-47D breast cancer cells.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B) is a key molecular driver inducing mutations in multiple human cancer. A3B belongs to the APOBEC3 enzyme family, which consists seven closely related DNA deaminases that catalyse cytosine-to-uracil (C>U) editing of single-stranded DNA (ssDNA). Using a cell model that lacks base excision repair function, we sampled A3B editing sites in bulk. Analyses conducted on these A3B editing sites point to a function of A3B in editing DNA:RNA hybrid structure known as R-loops. In order to verify this result, we conducted strand-specific DNA:RNA immunoprecipitation sequencing (ssDRIP-seq; S9.6, KeraFAST, ENH001) on T-47D breast cancer cells. Overall design: T-47D cells were divided into four sample groups and treated with siNT+DMSO, siNT+E2, siA3B+DMSO and siA3B+E2, where siNT and sA3B denote non-targeting and A3B-targeting siRNA respectively and E2 denotes 17-beta estradiol (E2). For each of the treatment group, three biological replicates were conducted, resulting a total of 12 samples. The effect of E2 and A3B knockdown on R-loop formation was investigated with quantified ssDRIP-seq.