ChIP-Seq in mouse embryonic stem cells of endogenous HA-tagged Tet1

R-loops are prevalent three-stranded nucleic acid structures, composed of a DNA:RNA hybrid and a displaced single-stranded DNA loop. Although aberrant R-loop formation is a threat to genome stability, R-loops are enriched at CpG islands (CGI) and are implicated in maintaining CGI promoters unmethylated. Mapping of genomic regions where Tet1 binds in an R-loop dependent manner we here identify almost exclusively CGIs. The results suggest that R-loops are genomic address labels for TET1. ChIP-Seq of endogenous HA-tagged Tet1 in E14TG2a mouse embryonic stem cells (mESHA-Tet1) was performed on permeabilized cells incubated with RNase H1 and Nuclease S1 or mock treated. Subsequently the cells were double crosslinked with disuccinimidyl glutarate (DSG) and formaldehyde. Fragmented chromatin was subjected to immunoprecipitation with HA antibody overnight.