Project description:Using high-throughput single-cell B-cell receptor sequencing (scBCR-seq) we obtained accurately paired full-length heavy- and light-chain variable regions from thousands of individual B cells in a massively parallel fashion. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion. In addition, we immunized rats with chicken ovalbumin and profiled antigen-reactive B cells from lymph nodes of immunized animals. The scBCR-seq data recovered 81% (n = 56/69) of B-cell lineages identified from hybridomas generated from the same set of B cells that were subjected to scBCR-seq. Importantly, scBCR-seq identified an additional 710 candidate lineages that were not recovered as hybridomas. We synthesized, expressed and tested 93 clones from the identified lineages and found that 99% (n = 92/93) of the clones were antigen-reactive. Our results establish scBCR-seq as a powerful tool for antibody discovery.
Project description:We have used RNA-seq to examine mRNAs from chicken spleen and bursa of Fabricius of three different condition (non-immunized and non-heat-stressed (24 ± 1℃ for 3 h), immunized (Newcastle disease vaccine) and non-heat-stressed, and immunized and heat-stressed (36 ± 1℃ for 3 h)). To clarify how chicken immune systems responded to heat stress with and without immunization.
Project description:The cell line DKO-S is derived form chicken B-Cell DT40 cells where the clathrin heavy-chain has been placed under the control of a tetracycline-regulatable promoter (Tet-Off). The originally derived cell-line DKO-S underwent apoptosis when clathrin expression was repressed. A cell line, DKO-R, derived from DKO-S cells that was less sensitive to clathrin-depletion. This project explores the differnces in gene expression between DKO-R and DKO-S cells
Project description:Chip-chip from pro-B cells from Rag1KO mice for H3K27ac and RNA Pol II Identification of novel enhancers from Ig heavy and light chain loci Rag1KO pro-B epigenetic landscape at Ig heavy and light chain loci
Project description:We developed an improved library prep protocol and standardized the data analysis pipeline for accurate repertoire profiling. In addition, two metrics were implemented to assess repertoire clone properties. We then studied systemically the effects of two adjuvants, CpG and Alum, on the Ig heavy chain repertoire using the ovalbumin (OVA) challenged mouse model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in terms of sequence mutation frequency, IGHV gene usage, CDR3 length, rescaled Hill numbers for clonal diversity, and clone selection strength. As a result, Ig repertoires of different tissues or isotypes exhibited distinguishable profiles at the non-immunized steady state. Adjuvanted immunizations further resulted in statistically significant alterations in Ig repertoire compared with PBS or OVA alone immunized groups. Lastly, we applied unsupervised machine learning techniques – multiple factor analysis and clustering – to identify Ig repertoire signatures in different compartments and under varying immunizations.
Project description:The cell line DKO-S is derived form chicken B-Cell DT40 cells where the clathrin heavy-chain has been placed under the control of a tetracycline-regulatable promoter (Tet-Off). The originally derived cell-line DKO-S underwent apoptosis when clathrin expression was repressed. A cell line, DKO-R, derived from DKO-S cells that was less sensitive to clathrin-depletion. This project explores the differnces in gene expression between DKO-R and DKO-S cells Duplicate samples ofDT40, DKO-S or DKO_R cells either with or without treatment with deoxycyclin are compared as is the expression between DKO-R and DKO-S cell lines
Project description:Chip-chip from pro-B cells from Rag1KO mice for H3K27ac and RNA Pol II Identification of novel enhancers from Ig heavy and light chain loci
