GSE183195
5C Chromatin interactions in the Igh locus in activated splenic B cells
These tracks include chromatin interaction data produced using the 5C (chromatin conformation capture carbon copy) method by the Kenter Lab located at the University of Illinois College of Medicine, Chicago, IL. These tracks show looping interactions across the Igh locus using alternating forward and reverse primer design scheme. The 3C method uses formaldehyde crosslinking to covalently associate interacting chromatin segments in intact cells. The cells are then lysed and chromatin is digested with the Hind III restriction enzyme. The digested fragments are ligated under dilute conditions to promote intra-molecular ligation. A genome-wide interaction library of ligation products corresponding to all possible chromatin interactions is produced by this method. In 5C assays, over 12,000 potential ligation products specific to the Igh locus are detected by PCR using multiplexed primer pairs. Here we focused on the 3' end of the Igh locus to evaluate the status of the constant region domain of the locus in LPS+IL4 activated splenic B cells.  We observe that the configuration of the 3' end of the Igh locus spanning the CH region genes assumes a developmental stage specific configuration in B lineage B cells. We find that the interaction frequency and activation state of the 3' enhancer increases as B cells mature. Overall design: The aim of this study was to create a comprehensive map of chromatin interactions spanning the entire 2.9 Mb Igh locus in LPS+IL4 activated splenic B cells. 5C primers follow an alternating pattern and were designed at HindIII restriction sites using the 5C primer design tools that are available online (http://my5C.umassmed.edu) (Lajoie et al., 2009) and were previously reported (Kumar et al., 2013).  In this experiment, 112 forward primers and 113 reverse primers were designed for all possible HindIII restriction fragments. This design allowed for the interrogation of many (but not all) restriction fragments, thus an unbiased interaction map across the locus and for potentially identifying new regulatory elements. An alternating scheme of forward and reverse primers was also included for a gene desert on chromosome 5 using 24 forward and 25 reverse 5C primers for a total of 525 potential cis interactions. Primers settings were: U-BLAST: 3; S-BLAST: 80: 15-MER: 800; MIN_FSIZE: 1000; MAX_FSIZE: 15,000; OPT_TM: 65; OPT_PSIZE: 30. Several primers with somewhat relaxed parameters were included to provide a more complete coverage of the Igh locus. Currently, data for two biological replicates have been generated for LPS+IL4 activated splenic B cells spanning the murine Igh locus and a gene desert on chromosome 5 using high-throughput paired-end sequencing in the Illumina GAII platform. The 5C looping interactions that are detected in both the biological replicates are considered significant.  These samples are part of a series of 5C analyses which were previously published (Montefiori et al. Cell Rep 2016 Feb 2;14(4):896-906).