Sequencing of Murine Somatic Hypermutation

Mice were immunised orally with sheep red blood cells for 2 weeks and intraperitoneally with 10 μg of LPS for 3 days. Single-cell suspensions from Peyer's patches were labelled with B220-APC- (Biolegend, ref:103212), GL7-FITC- (Beckton Dikinson, ref: 561530) and Fas-PE- (Beckton Dikinson, ref: 554258) conjugated antibodies. Purification of B220⁺GL7⁺Fas⁺ cells was realized on a FACS ARIA III (BD). Genomic DNA was extracted, and a region corresponding to a sequence of 517 bp downstream of the J_H4 segment was amplified by PCR. As a control, Igκ light-chain VJ-rearranged fragments were also amplified. Primers (detailed in the Supplementary Table 1) were coupled to 454 Sequencing adaptor sequences and PCR was performed using the program previously reported8 (link). According to the manufacturer, the resulting purified amplicons were prepared for sequencing with a GS Junior Titanium emPCR Kit (Lib-A; Roche), and the library of DNA fragments was sequenced on a 454 GS Junior instrument (Roche). Obtained sequences were aligned to the reference sequence using BWA aligner38 , and SAMtools software was used to obtain BAM files39 (link). Redundant sequences were excluded, and wig files were generated using IGV Tools40 (link) and manually analysed to determine mutation frequencies for each nucleotide in the sequence.