Igh repertoire analysis in primary pro-B cells from WT and NE1-/- mice (VDJ-seq)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA840929 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19334918_1.fastq.gz | Fastqsanger.gz | |||
| SRR19334918_2.fastq.gz | Fastqsanger.gz | |||
| SRR19334919_1.fastq.gz | Fastqsanger.gz | |||
| SRR19334919_2.fastq.gz | Fastqsanger.gz | |||
| SRR19334920_1.fastq.gz | Fastqsanger.gz |
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Project description:Analysis of the antibody repertoire composition is now possible using VDJ-seq. We used this recently developed method for unbiased amplification from genomic DNA (gDNA) to directly compare the Igh repertoire of C57Bl/6 (WT) and NE1-/- pro-B cells. We find that a group of contiguous proximal and intermediate VH genes are under-utilized in V->DJ rearrangement in the absence of NE1 revealing a NE1 zone of influence. We report the VH gene usage profile from WT and NE1-/- primary pro-B cells from the VDJ-seq data.
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2019-06-01 | GSE131090 | GEO
Project description:Rhesus macaques (RMs) are widely employed as a pre-clinical model in vaccination and infectious disease studies, yet their B cell immunobiology and immunogenetics remain ill-characterized. In this study, single-cell RNA/VDJ-seq (scRNA/VDJ-seq) was conducted on peripheral blood mononuclear cell (PBMC) samples from six RMs to describe the transcriptomic and V(D)JC repertoires of B cells and subtypes. 12 RM B cell clusters of distinct transcriptional states were identified, including IgM+ memory B cells (MBC), class-switched MBC, CD11c+ MBC and activated B cells. Novel gene signatures were also characterized for each B cell subtype, such as FCRL2 and CD24 for circulating marginal-zone-like B cells. In addition, VDJ repertoire properties of the global B cell population and each B cell subtype were elucidated, including IGH/K/L-V(D)JC gene family and subtype usage, class-switch recombination (CSR) status, somatic hypermutation (SHM) rate and levels, complementarity-determining region heavy chain (CDRH3) amino acid (AA) length and CDRH3 AA hydrophobicity scores. Interesting insights included the 1:1 ratio of kappa and lambda light chain usage and a preferential decreased IGHV3 but increased IGHV1 and 5 gene family usage in IGHG1 than IGHM-bearing B cells. Altogether, this study through comprehensive transcriptomic analyses identifies 12 distinct RM B cell subtypes paired with their respective V(D)JC repertoire, unraveling the complexity of B cell heterogeneity and improving future pre-clinical studies that can translate insights from this important non-human primate model to the understanding of human immunobiology.
2025-07-26 | GSE302169 | GEO
Project description:Purpose: IGH sequencing has revolutionized analysis of structural and functional signatures of B cell clonality. The goals of this study are to compare IGH repertoire of splenic B cells before and after knockdown of long isoform of PRLR. Methods: After amplifying the IGH repertoire of splenic B cells using PCR primers for VHJ558 variable and Cμ constant regions. For next generation sequencing (NGS), a TrueSeq library was prepared by adapter ligation to full-length products and sequenced on the MiSeq Illumina 2x150 bp platform. Results: In this project, Unique nucleotide sequences were identified, their abundances were calculated, and their complementary determining region 3 (CDR3) length, CDR3 spectrum, VDJ usage, and diversity were analyzed using the International Immunogenetics Information System (IMGT) HighV-QUEST software. Controls are included C3,C4,C5,C7,C8 and C9. LFPRLR SMO treated mice samples are: A3,A4,A5,A6,A7 and A8
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Project description:The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH- rearranged gene segment in the proximal Igh domain. By high-resolution mapping of long-range interactions, we now demonstrate that an array of local interaction domains establishes the three- dimensional structure of the extended Igh locus in lymphoid progenitors and thymocytes. In pro- B cells, these local domains engage in long-range interactions across the entire Igh locus, which depend on the transcription factors Pax5, YY1 and CTCF. The large VH gene cluster thereby undergoes flexible long-range interactions with the more rigidly structured 3M-bM-^@M-^Y proximal domain, which ensures that all VH genes can participate with similar probability in VH-DJH recombination to generate a diverse antibody repertoire. Notably, these long-range interactions appear to be an intrinsic feature of the VH gene cluster, as they are still generated upon mutation of the EM-NM-< enhancer, IGCR1 insulator or 3M-bM-^@M-^Y regulatory region present in the 3M-bM-^@M-^Y proximal Igh domain. 4C sequencing from mutliple celltypes with multiple viewpoints; uneven number of replicates ChIP-Seq
2013-09-30 | E-GEOD-43008 | biostudies-arrayexpress
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5M-bM-^@M-^Y-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. For the ChIP-seq, input and immunoprecipitated DNA was given to The Scripps DNA Array Facility, where it was prepared for massively parallel sequencing on Illumina HiSeq2000.
2013-08-05 | E-GEOD-47766 | biostudies-arrayexpress
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5M-bM-^@M-^Y-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. RNA was extracted from C57BL/6 RAG-/- pro-B cells using TrizolM-BM-. (Life Technologies Corp., Carlsbad CA) and genomic DNA was eliminated using the genomic DNA wipeout buffer in the QuantiTect Reverse transcription kit (QIAGEN). A final purification of the RNA was performed with the RNeasy kit from QIAGEN. For each sample, 100 ng of total RNA was used to make RNASeq libraries using the NuGEN Encore Complete DR kits following manufacturer's recommended protocols. Sequencing libraries were gel purified to ensure insert sizes were larger than 100 bp in length and sequenced on an Ilumina HiSeq2000 for 100 bases plus 7 bases for indexing.
2013-08-05 | E-GEOD-47885 | biostudies-arrayexpress
Project description:The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH- rearranged gene segment in the proximal Igh domain. By high-resolution mapping of long-range interactions, we now demonstrate that an array of local interaction domains establishes the three- dimensional structure of the extended Igh locus in lymphoid progenitors and thymocytes. In pro- B cells, these local domains engage in long-range interactions across the entire Igh locus, which depend on the transcription factors Pax5, YY1 and CTCF. The large VH gene cluster thereby undergoes flexible long-range interactions with the more rigidly structured 3’ proximal domain, which ensures that all VH genes can participate with similar probability in VH-DJH recombination to generate a diverse antibody repertoire. Notably, these long-range interactions appear to be an intrinsic feature of the VH gene cluster, as they are still generated upon mutation of the Eμ enhancer, IGCR1 insulator or 3’ regulatory region present in the 3’ proximal Igh domain.
2013-09-30 | GSE43008 | GEO
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5’-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus.
2013-08-05 | GSE47766 | GEO