title: GSM1145567: IgH_FB_V_reg_22552_BL6_distalV; Mus musculus; OTHER
library_construction_protocol: The initial steps of the 4C procedure, as published before (Simonis et al., 2006, Nature Genetics), remain unchanged. Cells are cross linked using 2% formaldehyde for 10min at room temperature in PBS 10%FCS, nuclei are isolated, after which chromatin is digested with HindIII and subsequently ligated under diluted conditions. After reversal of the cross links the DNA is purified and treated with the second restriction enzyme, DpnII. After a second re-ligation step the sample is purified and a 4C PCR is applied. FACS analysis is used to separate the cells on the basis of the allotypic origin of the productive IgH allele. The FVB allele codes for the B cell receptor from the IgMa isotype, whereas the c57Bl/6 allele encodes for the B cell receptor from the IgMb isotype. Allele specific analysis is enabled applying paired end sequencingon the sorted IgMa and IgMb cell populations. One read (P1) reads the 4C capture, whereas the other read (P2) identifies the genotype of the mouse and thus the productive or non-productive allele. Illumina adaptor attachment is done by extending the 4C PCR primers with the paired-end Illumina adaptors. These extended 4C PCR primers are used in the 4C PCR protocol