Mapping and quantification of single-strand DNA bound by flag-tagged APOBEC3B

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B, A3B) 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). Here, we adopted single-strand DNA protein-immunoprecipitation sequencing (SPI-seq) to capture the ssDNA bound by flag-tagged A3B protein. Quantification on sequence reads was carried out to investigate the effect of 17-beta estradiol (E2) on A3B binding to DNA. T-47D breast cancer cells transduced with a lentiviral vector that allows inducible expression of flag-tagged APOBEC3B protein. After induction of the lentiviral APOBEC3B cassette by doxycycline, SPI-seq experiments were carried out using T-47D cells treated with DMSO or E2 (anti-flag antibody (M2, Sigma, F1804)) Quantification was carried out using n = 2 biological repeats to investigate the effect of E2 stimulation on A3B binding.