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Series GSE165776 Query DataSets for GSE165776
Status Public on Jun 01, 2022
Title Next Generation Sequencing Identifies Biased Light Chain Usage and Evidence of Light Chain Gene Replacement in phosphatidylcholine (PtC)-specific CD5+ B cells from dnRAG1 and VH12 mice.
Organism Mus musculus
Experiment type Other
Summary B cells reactive toward phosphatidylcholine (PtC) are enriched in the B1 B cell subset, and express predominantly one of two VH/Vk combinations to confer this specificity: VH12/Vk4/5H and VH11/Vk9. Studies of transgenic mice expressing the VH12 heavy chain (VH12 mice) suggest two major checkpoints for light chain expression in this system: the first involves selection of V-J rearrangements which encode a “permissive” light chain that can functionally pair with the VH12 heavy chain; the second involves receptor editing to salvage non-PtC reactive B cells to acquire a permissive light chain that confers PtC reactivity. If this model is correct, impairing receptor editing should reduce the frequency of PtC-reactive B1 B cells in VH12 mice. To test this possibility, we bred VH12 mice to transgenic mice expressing a catalytically inactive form of RAG1 (dnRAG1 mice) which show a defect in receptor editing. Interestingly, dnRAG1 expression in VH12 mice enforces development of PtC-reactive B1 B cells, rescuing the loss of splenic B cells observed in VH12 mice. These data suggest receptor editing normally functions to remove a large portion of PtC-specific B cells in VH12 mice. Deep sequencing of the expressed light chain repertoire of PtC-reactive and non-reactive B cells in VH12 mice revealed that PtC-reactive B cells predominantly expressed the Vk4/5H (IGKV4-91) light chain gene, whereas PtC-non-reactive B cells expressed a broader, yet restricted, set of light chain genes. This analysis also revealed a low frequency of in-frame hybrid light chain genes appearing to originate via Type 2 gene replacement, which we show can originate from template switching during PCR.
Overall design The sequencing libraries were generated from total RNA extracted from 60 samples. 24 samples were analyzed in the first round, these were comprised of 3 independent biological replicates for 8 unqiue cell populations from the spleens of 4 geneotypes of mice. For the second round 36 samples were anlysed, comprised of 3 independent biological replicates for 12 unique cell populations from the bone marrow and spleen of 2 genotypes of mice.
Contributor(s) Worth AN, Palmer VL, Schabla NM, Perry GA, Fraser-Philbin AN, Swanson PC
Citation(s) 35705027
Submission date Jan 29, 2021
Last update date Aug 31, 2022
Contact name Patrick Swanson
Organization name Creighton University School of Medicine
Department Medical Microbiology and Immunology
Lab Swanson
Street address 2500 California Plaza
City Omaha
State/province NE
ZIP/Postal code 68178
Country USA
Platforms (1)
GPL16417 Illumina MiSeq (Mus musculus)
Samples (60)
GSM5049729 236_5N_LN
GSM5049730 237_5N_LN
GSM5049731 237_5P_LN
BioProject PRJNA697869
SRA SRP303817

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Supplementary file Size Download File type/resource
GSE165776_RAW.tar 34.4 Mb (http)(custom) TAR (of TAB)
GSE165776_V-QUEST_files.tar.gz 3.9 Mb (ftp)(http) TAR
GSE165776_full_graph_files.tar.gz 95.0 Kb (ftp)(http) TAR
GSE165776_seq_logo_files.tar.gz 1.4 Mb (ftp)(http) TAR
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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