Mapping the genomic occupancy by flag-tagged APOBEC3B protein

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B) is a key molecular driver inducing mutations in multiple human cancers. APOBEC3B 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). In order to investigate the genomic binding sites of APOBEC3B, high through-put sequencing experiments were conducted using DNA samples acquired by standard chromatin immunoprecipitation (ChIP) and ssDNA protein immunoprecipitation (SPI) samples. Substrate preference by APOBEC3B were characterised by comparing these results. Overall design: 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, cells were transfected with plasmid that allow transient expression of human RNase H, or treated with transfection agent as control. Treated cells were subjected to ChIP-seq and SPI-seq with anti-flag antibody (M2, Sigma, F1804), which detect APOBEC3B-bound dsDNA or ssDNA respectively.