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SRX7199800: GSM4185198: NP10d-3_SP_IgH_nonGC; Mus musculus; OTHER
1 ILLUMINA (Illumina MiSeq) run: 352,565 spots, 212.2M bases, 132.2Mb downloads

Submitted by: NCBI (GEO)
Study: Peyer's Patch Germinal Centers Select Innate-like BCRs
show Abstracthide Abstract
Primary B cell receptor (BCR)/antibody variable region exons are generated by V(D)J recombination with junctional diversification creating immensely diverse antigen contact-encoding CDR3s. While most antigen-driven germinal centers (GCs) are transient, gut microbiota-dependent intestinal Peyer's patch (PP) GCs are chronic. The nature of chronic PP GC BCR repertoires and somatic hyper-mutation (SHM) patterns has remained enigmatic. To elucidate the physiological repertoire of PP GC BCRs, we developed a high throughput antibody repertoire and SHM assay. Remarkably, PP GCs from different mice expanded public clonotypes, each often with identical IgH CDR3. These CDR3s represent innate-like BCRs that appear much more frequently than expected in naïve B cell repertoire by IGoR modeling, but not frequently enough to enter PP GCs at the observed recurrence without cellular selection. Consistently, some public clonotypes are gut microbiota-dependent and encode antibodies reactive to bacteria glycans, while others are not. SPF fecal transfer to germ-free (GF) mice restored two germ-dependent clonotypes, providing direct evidence for BCR selection. In support of this, we identified recurrently selected SHMs in four of the public clonotypes, demonstrating affinity maturation in chronic PP GCs. Our findings suggest that persistent gut antigens select for innate-like BCR clonotypes to seed chronic PP GCs. Overall design: We validated our primer design for LAM-HTGTS-Rep-SHM-Seq with mixed JH or JL bait compared with VH or VL bait, with 3 replicates each. We included NP-CGG and SRBCs IP immunized splenic GC and naïve samples as controls for our LAM-HTGTS-Rep-SHM-Seq method to follow BCR selection. We did LAM-HTGTS-Rep-SHM-Seq on PP GC and naïve samples from 18 SPF mice, 10x3 GF mice, 5 GF+SFB mice, 10 GF+SPF mice, 15 AID-deficient SPF mice. We also included single cell RNA sequencing data from 4 of the PP GC samples from SPF mice.
Sample: NP10d-3_SP_IgH_nonGC
SAMN13348245 • SRS5705355 • All experiments • All runs
Organism: Mus musculus
Library:
Instrument: Illumina MiSeq
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: 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.
Experiment attributes:
GEO Accession: GSM4185198
Links:
Runs: 1 run, 352,565 spots, 212.2M bases, 132.2Mb
Run# of Spots# of BasesSizePublished
SRR10511612352,565212.2M132.2Mb2020-02-22

ID:
9455946

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