title: END-seq-preB-RAG1-1
design: Single cell suspensions were washed in PBS, embedded in agarose plugs and processed for END-seq (detailled in the manuscript). Agarose plugs were treated with proteinase K and RNase A. After blunting and A-tailing the DNA ends were captured by ligation with a biotinylated hairpin adapter containing a 3’T overhang and Illumina’s p5 sequence. Subsequently, the agarose plug was melted, DNA was extracted from the plug and sheared, after which fragments containing the adapter (and the DNA end) were captured with streptavidin-coated beads. The new ends created by sonication were also end-repaired and A-tailed, allowing ligation of a second hairpin adapter containing the Illumina’s p7 sequence. PCR amplification with Illumina TruSeq PCR primers for 16 cycles resulted in a ready-to-sequence library in which the first base sequenced (read 1) corresponded to the first base of the blunted DSBs. Control input libraries from thymocytes, pre-B cells or B cells were generated using 5 ng of sonicated DNA, and processed as detailed above in the second adapter processing step, with the exception of the streptavidin purification. Control libraries were prepared using Illumina TruSeq universal adapters and 15 cycles of PCR. All libraries were quantified using Picogreen or quantitative PCR. Sequencing was performed either on the Illumina HiSeq (50bp single end reads) or on the Illumina NextSeq500 (75bp single end reads).