3 RAG1/2 introduces double-stranded breaks at non-Ig loci | 59 out of the gel and to perform nick translation on non-ligated 3'-ends of DNA fragments. Finally, the libraries were amplified using Platinum® PCR amplification kit and the final size distribution was checked using Agilent Technologies 2100 Bioanalyzer™. To sequence the libraries an amplification on P1 beads was performed in an emulsion PCR (ePCR), followed by enrichment of the templated beads. Templated bead preparation was performed using the SOLiD® EZ Bead™ System (Applied Biosystems) according to the manufacturers’ protocol. The emulsion was dispensed into a 96-well plate and cycled for 60 cycles. After the amplification, emulsion was broken with butanol, beads were enriched for template positive beads, 3'-end extended and covalently attached to sequencing slides. Physically separated samples were deposited on one sequencing slide and sequenced using standard settings on the 5500 Series SOLiDTM sequencer. Analysis of ChIP-Seq data Color signal/SOLiD signal was base-called into reads with the manufacturer’s software (Lifescope). Sequencing reads were quality trimmed by clipping at 3 consecutive nucleotides with a quality score of less than 10. Reads shorter than 18 nucleotides were discarded and the remaining reads were cleaned of PCR duplicates and mapped onto the mouse genome (MM9 assembly) using the Bowtie24 tool available on the Galaxy platform. NBS1 peak calling was performed by MACS25 in each cell line. Next, to identify the RAG-dependent NBS1 peaks, the peaks called in Artemis-/-/Rag2-/- (R2K.Art) were subtracted from breaks called in the RAG proficient Artemis-/- cell (AH2). Finally, the peaks called in the Input sample were subtracted from the RAG-dependent NBS1 peaks to eliminate any background binding bias. Annotation of the resulting RAG-dependent NBS1 peaks cleaned of input background was performed using PAVIS algorithm26 for which the RAG-dependent NBS1 peaks were first lifted over to MM10 assembly. The unsupervised motif analysis was performed by STREME (Sensitive, Thorough, Rapid, Enrichment Motif Elicitation)27. The program uses Fisher’s Exact Test or the Binomial test to determine the significance of each motif found in the positive set as compared with its representation in the control set. Shuffled primary sequences were used as a control set. The identified motifs were then compared to known motifs using the Tomtom motif comparison tool28. This tool compares the identified motifs against the database of known motifs, produces alignment for each identified motif, and assigns a rank. The supervised analysis of the simple repeat motif enrichment was performed by SEA (Simple Enrichment Analysis)29, which compares the used-provided motifs in the primary dataset and compares them against the enrichment in the shuffled primary sequences used as a control data set. The cut-off for q-value, the minimal false discovery rate at which the observed similarity would be deemed significant, was set to 0.05 in all motif-finding analyses.
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