Isolation of repaired chromatin of UVC-irradiated human skin fibroblasts

To further study and understand the DNA damage responses, we developed aniFOUND, a new method for capturing under native conditions the repaired DNA and the proteins associated with it after UVC irradiation. hTert-immortalised human skin fibroblasts were irradiated with 20 J/m2 UVC and the nascent DNA that resulted from the Unscheduled DNA Synthesis was specifically labeled with nucleotide analogues (EdU). This newly repaired/synthesized DNA together with the bound proteins were pulled-down with streptavidin beads. For negative controls we used both irradiated, but non-labelled samples (+UV/-EdU) and labelled, but not irradiated cells (-UV/+EdU). The pulldowns were eluted from the beads by boiling in 0.1 % SDS.