HEK293T cells were transfected with p84-ZFN or other ZFNs and allowed to recover for 18 hr. Cells were fixed in 1% methanol-free formaldehyde for 10 min to crosslink proteins, lysed in ChIP buffer (Cell Signaling Technology, MA, USA), sonicated, and cleared by centrifugation. Part of the supernatant was digested with proteinase K (65 °C for 2 hr), the DNA isolated by spin columns and input DNA quantitated by Real Time PCR. Equivalent amounts of chromatin were incubated with primary antibody (overnight at 4 °C) followed by protein G agarose beads precoated with sperm DNA. Immune complexes were washed in low and high salt ChIP buffers (Cell Signaling Technology, MA), eluted, incubated in NaCl (65 °C for 2 hrs) and digested with proteinase K. Purified DNA was quantitated by RT-qPCR using the Step One Plus real time PCR system (Applied Biosystems, CA). PCR protocols, primer pairs and ChIP grade antibodies are listed in supplementary methods.
DSB production was monitored using standard PCR based techniques previously described by us10 (link). Genomic DNA prepared for ChIP was amplified using primer pairs located either side of the DSB (supplementary methods) by real time qPCR and the percent of DSBs estimated by the change in signal resulting from cleavage of the DNA. 18s rRNA genomic DNA signal was used to ensure equal input DNA.
DSB production was monitored using standard PCR based techniques previously described by us10 (link). Genomic DNA prepared for ChIP was amplified using primer pairs located either side of the DSB (supplementary methods) by real time qPCR and the percent of DSBs estimated by the change in signal resulting from cleavage of the DNA. 18s rRNA genomic DNA signal was used to ensure equal input DNA.
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