Project description:Present R loop profling oftern requires large amout of material, lacking information about R loop in primary cells. To overcome this setback, we delevopment low input method which integrates in vitro amplication capability of T7 promoter and high binding specificity between nanotag and IgH (mouse)
Project description:Present R loop profling oftern requires large amout of material, lacking information about R loop in primary cells. To overcome this setback, we delevopment low input method which integrates in vitro amplication capability of T7 promoter and high binding specificity between nanotag and IgH (mouse)
Project description:Present R loop profling oftern requires large amout of material, lacking information about R loop in primary cells. To overcome this setback, we delevopment low input method which integrates in vitro amplication capability of T7 promoter and high binding specificity between nanotag and IgH (mouse)
Project description:Present R loop profling oftern requires large amout of material, lacking information about R loop in primary cells. To overcome this setback, we delevopment low input method which integrates in vitro amplication capability of T7 promoter and high binding specificity between nanotag and IgH (mouse)
Project description:Probing histone modifications at a single-cell level in thousands of cells has been enabled by technologies such as single-cell CUT&Tag. Here we describe nano-CUT&Tag (nano-CT), which allows simultaneous mapping of up to three epigenomic modalities at single-cell resolution using nanobody-Tn5 fusion proteins. Multimodal nano-CT is compatible with starting materials as low as 25,000-200,000 cells and has significantly higher sensitivity and number of fragments per cell than single-cell CUT&Tag. We use nano-CT to simultaneously profile chromatin accessibility, H3K27ac, and H3K27me3 in juvenile mouse brain, allowing for discrimination of more cell types and states than unimodal single-cell CUT&Tag. We also infer chromatin velocity between assay for transposase-accessible chromatin (ATAC) and H3K27ac in the oligodendrocyte lineage and deconvolute H3K27me3 repressive states, finding two sequential waves of H3K27me3 repression at distinct gene modules during oligodendrocyte lineage progression. Given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in complex biological systems at the single-cell level.
Project description:R-loops are ubiquitous, dynamic nucleic-acid structures that play fundamental roles in DNA replication and repair, chromatin and transcription regulation, as well as telomere maintenance. The DNA-RNA hybrid-specific S9.6 monoclonal antibody is widely used to map R-loops. Here, we report crystal structures of a S9.6 antigen-binding fragment (Fab) free and bound to a 13-bp hybrid duplex. We demonstrate that S9.6 exhibits robust selectivity in binding hybrids over double-stranded (ds) RNA and in categorically rejecting dsDNA. S9.6 asymmetrically recognizes a compact epitope of two consecutive RNA nucleotides via their 2'-hydroxyl groups and six consecutive DNA nucleotides via their backbone phosphate and deoxyribose groups. Recognition is mediated principally by aromatic and basic residues of the S9.6 heavy chain, which closely track the curvature of the hybrid minor groove. These findings reveal the molecular basis for S9.6 recognition of R-loops, detail its binding specificity, identify a new hybrid-recognition strategy, and provide a framework for S9.6 protein engineering.
