ice-cold PBS. Cells were lysed by incubating for 5 mins in cold lysis buffer. Resulting nuclei were washed in MNase buffer. Chromatin was subjected to micrococcal nuclease digestion, with pre-determined MNase concentration, in MNase buffer at RT to yield nucleosomal fragments with less than 3 nucleosomes. 25 mM EDTA and 0.2% SDS was used to stop the reaction and sample was centrifuged to remove cellular debris. Lysates were diluted 1:10 in ChIP dilution buffer. Non-specific background was removed by incubating the MNase digested chromatin with dynabeads overnight at 4C with rotation in the presence of BSA. Precleared chromatin solutions were incubated overnight at 4C with rotation with antibodies against PARP1 and for control, IgG. The immuno-complexes were collected with 200 l of MagnaChIP protein A beads for 3 h at 4C with rotation. The beads were washed sequentially for 3 min by rotation with 1 ml of the following buffers: low salt wash buffer, high salt wash buffer and LiCl wash buffer. Finally, the beads were washed twice with 1 ml TE buffer. The 
immuno-complexes were then order EMA-401 eluted by adding 500 l of elution buffer and incubating for 60 min on rotation. The cross-linking was reversed at 65C and the remaining proteins were digested with 80 g/ml proteinase K and incubating overnight at 65C. The DNA was recovered by purifying with Qiagen PCR purification kit. Immunoprecipitated chromatin DNA was then run on a 3.3% Nusieve gel. Mononucleosomal fragments were cut from the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723429 gel and adapted for deep-sequencing as we described previously. For ChIP-qPCR experiments, eluted samples were used for quantitative real-time PCR or normal PCR. For MNase-seq analyses, experiments were performed as we had previously done. Preparation of ChIP-seq libraries Sequencing libraries were prepared with the SOLiD ABI technologies as previously described. Briefly: ChIPped mononucleosomal DNA was gel purified, ligated with the adapters and 3 / 22 Functional Location of PARP1-Chromatin Binding then PCR amplified with SOLiD primers for 10 cycles. PCR product, corresponding to mononucleosomal DNA, was gel purified and loaded onto a SOLiD flow cell for cluster generation and genome-wide sequencing. Data Analyses PARP1 colorspace fasta reads were converted to basespace fastq format by the Northwestern University Genomics core using a custom script. We sorted PARP1 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723632 reads into separate library files based on their barcodes, and mapped the reads to the human genome using bowtie v2 with the default parameters allowing for two mismatches. Unique reads with zero or one mismatch were considered for further analysis. Amplification bias was removed by filtering more than two copies of redundant reads that come from the same locus. We compared how well technical replicates within each cell of PARP1 binding overlapped each other. The degree of overlap among peak-sets for all two biological replicates was more than 75%, demonstrating a high level of reproducibility further confirmed with a Pearson correlation of r > 0.823; p< 10-16. Because of this high reproducibility between the replicates, the unique reads were combined and reanalyzed to produce composite binding regions. A total of 54,125,493 and 60,486,178 non-redundant uniquely mapped reads were obtained for MCF7 and MDA-MB231 cells respectively. Defining PARP1 binding sites. Two different methods were used to analyze PARP1 binding regions since no peak-calling program has gained consensus acceptance by the scientific commun