Chip buffer

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 us^{10 (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.

MT/c-Src^+/+ cells in 15 cm plates (Nunc, 168381) were fixed in 1% formaldehyde for 10 minutes, and the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9003) was used to prepare chromatin according to the manufacturer’s protocol. Equal amounts of digested, cross-linked chromatin were diluted in 1× ChIP Buffer (Cell Signaling Technology, 7008) and incubated with antibodies against FOXM1 (Proteintech, sc-376471, 1:100), Histone H3 (Cell Signaling Technology, 4620, 1:50), or normal rabbit IgG (Cell Signaling Technology, 2729, 1:500) overnight at 4°C with end-over-end mixing, and then incubated with ChIP-Grade Protein G Magnetic Beads (Cell Signaling Technology, 9006) for 2 hours at 4°C with end-over-end mixing. Beads were washed 4 times with ChIP Buffer, and DNA was eluted with 50 μL DNA Elution Buffer (Cell Signaling Technology, 10009). ChIP enrichment was quantified by qRT-PCR as described above (primer details are provided in Supplemental Table 5). CCNB1 and PLK1 were used as positive controls and ACTB and RPL30 as negative controls for FOXM1 binding.

HEK293T cells were transfected with p84-ZFN to create a specific DSB in the PPP1R12C gene. 18hr later, cells were fixed (1% methanol-free formaldehyde/10 min) to crosslink DNA and proteins, then lysed in Chromatin Immunoprecipitation (ChIP) buffer using kits (Cell Signaling Technology, MA, USA: #9005). Samples were then 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 (using primers against 18S rRNA genomic sequence). For ChIP, equivalent amounts of chromatin were incubated with H4Ac antibody (Millipore, CA: 06-866) 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 - #9005), eluted, incubated in NaCl (65°C for 2 hrs) and then 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 and primer pairs are listed below.