Project description:SPO11-promoted DNA double-strand breaks (DSBs) formation is a crucial step for meiotic recombination, and it is indispensable to detect the broken DNA ends accurately for dissecting the molecular mechanisms behind. Here, we report a novel technique, named DEtail-seq (DNA End tailing followed by sequencing), that can directly and quantitatively capture the meiotic DSB 3’ overhang hotspots at single-nucleotide resolution.
Project description:The chemokines CXCL13 and CXCL12 are reported to be important for the germinal center reaction. Since CXCL12-deficient mice are embryonically lethal, here we took advantage of the Cxcl13-Cre/TdTomato mouse models to genetically ablate CXCL12 from B cell-interacting reticular cells and examine the molecular consequence on germinal center B cells. Spatial segregation of follicular dendritic cells, germinal center B cells and follicular helper T cells is impaired in Cxcl13-Cre/TdTomato Cxcl12fl/fl mice. Single cell transcriptomic analysis revealed that all germinal center B cell subsets (corresponding to distinct stages of the germinal center response) are present in draining lymph nodes of immunized CXCL12-conditionally deficient mice. While most transcriptional regulators of the germinal center response are unperturbed by the genetic perturbation of CXCL12, Bach2 levels were elevated in germinal center B cells from lymph nodes of Cxcl12fl/fl mice. Moreover, single cell B cell receptor sequencing revealed that germinal center B cells in Cxcl13-Cre/TdTomato Cxcl12fl/fl mice harbour a lower mutational burden when compared to germinal center B cells isolated from immunized control mice. Gene expression profiles were validated by flow cytometry and suggest that the provision of CXCL12 by reticular cells governs efficient germinal center responses.
