CUT&TAG analysis of R-loops in Mcm8 knockout MEFs and CUT&TAG analysis of R-loops and MCM8-binding sites in HEK293 cells
To reveal the role of MCM8 in suppressing R-loop accumulation, we performed the CUT&TAG assay using the S9.6 antibody to map genome-wide R-loops in Mcm8 wildtype MEFs and Mcm8 knockout MEFs. We also conducted the CUT&TAG assay to detect genome-wide R-loops in Ddx5 downregulated MEFs by adenovirus infection and in control MEFs. To investigate the underlying molecular mechanism of MCM8 suppressing R-loops, we conducted the DNA sequencing of libraries from CUT&TAG assay using the antibody against FLAG in HEK293 cells transfected with FLAG-MCM8 plasmid and using the S9.6 antibody in HEK293 cells. Besides, an IgG control and control of RNH1 overexpression were included. Overall design: CUT&TAG-seq for R-loops using the S9.6 antibody in Mcm8 wildtype and MCM8 knockout MEFs. CUT&TAG-seq for R-loops using the S9.6 antibody in DDX5 downregulated MEFs and control MEFs. CUT&TAG-seq for R-loops using the S9.6 antibody in HEK293 cells with or without RNH1 overexpression.  CUT&TAG-seq for MCM8-binding sites using the anti-FLAG antibody in HEK293 cells with or without FLAG-MCM8 overexpression.CUT&TAG-seq using the no-specific antibody IgG was used as a control.