GSM4185194: NP10d-1_SP_IgH_nonGC; Mus musculus; OTHER
Spleen and PPs were dissected out from 8-12 week old mice, prepared into single cell suspensions and purified by EasySep® Negative Selection B cell Enrichment Kit (Stem Cell Techonologies) according to the manufacturer's protocol. Purified B cells were stained with anti-B220-PE (1:2000) (eBiosciences), anti-CD38-APC (1:200) (eBiosciences) and anti-GL7-FITC (1:200) (Biolegend). GC (B220+GL7+CD38-) and nonGC (B220+GL7-CD38+) B cells were sorted from the same sample by fluorescence-activated cell sorting (FACS). Genomic DNA from sorted cells was prepared using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's protocol. LAM-HTGTS-Rep-SHM-Seq was derived from HTGTS-Rep-seq (Lin et. al., PNAS 2016) with several modification. All libraries were made using 400 ng genomic DNA as starting materials, so that the level of PCR amplification was kept the same among different samples. In the cases when less than 400 ng genomic DNA was obtained from GC or non-GC B cell samples, genomic DNA from JM8a3, a C57BL/6 ES cell line, was added to make the starting DNA template amount consistent among samples. The library preparation procedure was mostly done as previously described with a few modifications in the linear amplification-mediated PCR step, where we used one reaction of 50 μl instead of 8 reactions and performed 100 cycles instead of 80 cycles. scRNA-seq libraries were prepared using 10x Genomics Library Kits and sequenced on an Illumina NextSeq500 according the manufacturer's recommendations.