Sequencing Sμ Mutations in Activated B Cells

Analysis of mutations at Sμ by NGS was performed as described in Pérez-Durán P et al.^{51 (link)}. Briefly, naive B cells were purified by immunomagnetic depletion with anti-CD43 beads (Miltenyi) and cultured with 25 μg/ml LPS (Sigma) plus 10 ng/ml IL4 (PeproTech) for 3 days to obtain activated B cells. Immature B-cell populations were purified by FACs-sorting as follows: B220+ CD19−, pre-pro-B; B220+ CD19+ IgM-CD25− pro-B; B220+ CD19+ IgM-CD25+ pre-B; B220+ CD19+ IgM+ IgD−, immature. Genomic DNA from purified B-cell populations was extracted, and PCR amplified with Pfu Ultra high-fidelity DNA polymerase (Stratagene). PCR reactions were fragmented with Covaris, and adapter-ligated libraries were generated, processed, and sequenced on a HiSeq2500 according to the manufacturer’s instructions (Illumina).