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SRX3466739: Mus musculus: Swiss webster, Igh conventionalized for 21days at day21-22: Antibody Heavy Chain Sequences from Lamina propriar B cells
1 ILLUMINA (Illumina MiSeq) run: 113,554 spots, 68.1M bases, 36.9Mb downloads

Design: LP B cells: DAPI-CD19+B220+IgM+CD93-CD95-GL7-CD23+ were sorted from conventionalized SW (the conventionalization was initiated in d21- d22-old SW GF littermates for 21 days). Total RNA was extracted from sorted B cells using TRIzol reagents. The RNA was reverse transcribed by Superscript III reverse transcriptase with Oligo dT primer. The first-strand cDNA was purified by the Agencourt RNAClean XP beads to remove the primers. 19 forward degenerate primers binding to the framework region 1 of the VH region barcoded by a unique 8 nucleotide molecular identifier (UMI) and connected with the universal primer were used for the second strand cDNA synthesis and barcoding. After removing the residual primers by RNAClean XP, the barcoded cDNA was amplified for 25 cycles by constant region primer and universal primer to add 5 and 3 adaptors. The PCR products were purified by QIAquick PCR purification kit, and amplified again for 18 cycles to add Illumina linkers (P5 and P7) and sample barcodes. Products were quantified on a Qubit fluorimeter, run on a fragment analyzer with amplicon regions and expected sizes confirmed. Samples were then pooled in equal amounts based on product concentration. The pooled products were then size selected on an agarose gel and purified by gel extraction. Purified, size-selected products were run on an Agilent Bioanalyzer to confirm appropriate profile and determination of average size. The final pools were quantitated using Qubit and diluted to 5nM final concentration. The 5nM dilutions were further quantitated by qPCR on a BioRad CFX Connect Real-Time System and pooled evenly. The pool was denatured and spiked with 15% non-indexed PhiX control library provided by Illumina and loaded onto the MiSeq v3 flowcell at a concentration of 7 pM for cluster formation and sequencing. The PhiX control library provides a balanced genome for calculation of matrix, phasing and prephasing, which are essential for accurate base calling. The libraries were sequenced from both ends of the molecules to a total read length of 250nt from each end.
Submitted by: Brigham and Women's Hospial and Harvard Medical School
Study: Microbial symbionts regulate the primary Ig repertoire
show Abstracthide Abstract
The ability of immunoglobulin (Ig) to recognize pathogens is critical for optimal immune fitness. Early events that shape pre-immune Ig repertoires, expressed on IgM+ IgD+ B cells as B cell receptors (BCRs), are poorly defined. Here we studied germ-free mice and conventionalized littermates to explore the hypothesis that symbiotic microbes help shape the pre-immune Ig repertoire. Ig binding assays showed that exposure to conventional microbial symbionts enriched frequencies of anti-bacterial IgM+ IgD+ B cells in intestine and spleen. This enrichment affected follicular B cells, involving a diverse set of Ig variable region gene segments, and was T cell-independent. Functionally, enrichment of microbe reactivity primed basal levels of small intestinal T cell-independent symbiont-reactive IgA, and enhanced systemic IgG responses to bacterial immunization. These results demonstrate that microbial symbionts influence host immunity by enriching frequencies of anti-bacterial specificities within pre- immune B cell repertoires.
Sample: Mus musculus: Swiss webster, Igh conventionalized for 21days at day21-22: Antibody Heavy Chain Sequences from Lamina propriar B cells
SAMN08167475 • SRS2754512 • All experiments • All runs
Organism: Mus musculus
Name: LP_Col_Day21_YC214_12
Instrument: Illumina MiSeq
Strategy: AMPLICON
Selection: PCR
Layout: PAIRED
Runs: 1 run, 113,554 spots, 68.1M bases, 36.9Mb
Run# of Spots# of BasesSizePublished


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