Nanobody-tethered Transposition Allows for Multifactorial Chromatin Profiling at Single-cell Resolution
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PROVIDER: PRJNA884478 | ENA |
REPOSITORIES: ENA
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Project description:Nanobody-tethered Transposition Allows for Multifactorial Chromatin Profiling at Single-cell Resolution
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Project description:Nanobody-tethered transposition enables multifactorial chromatin profiling at single-cell resolution
| PRJNA876138 | ENA
Project description:Chromatin states are functionally defined by a complex combination of histone modifications, transcription factor binding, DNA accessibility, and other factors. However, most current single-cell-resolution methods are unable to measure more than one aspect of chromatin state in a single experiment, limiting our ability to accurately measure chromatin states. Here, we introduce nanobody-tethered transposition followed by sequencing (NTT-seq), a new assay capable of measuring the genome-wide presence of multiple histone modifications and protein-DNA binding sites at single-cell resolution. NTT-seq utilizes recombinant Tn5 transposase fused to a set of secondary nanobodies (nb). Each nb-Tn5 fusion protein specifically binds to different immunoglobulin-G antibodies, enabling a mixture of primary antibodies binding different epitopes to be used in a single experiment. We apply bulk- and single-cell NTT-seq to generate high-resolution multimodal maps of chromatin states in cell culture and cells of the human immune system, demonstrating the high accuracy and sensitivity of the method. We further extend NTT-seq to enable simultaneous profiling of cell-surface protein expression alongside multimodal chromatin states to study cells of the immune system.
2022-09-21 | GSE212588 | GEO
Project description:Multifactorial stress combination
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Project description:A nanobody is an antibody fragment consisting of a single monomeric variable antigen-binding domain. Mammalian cells are ideal platforms for identifying nanobodies targeting hard-to-display transmembrane proteins and nanobodies that function as modulators of cellular phenotypes. However, the introduction of a high-diversity nanobody library into mammalian cells is challenging. We have developed two novel methods for constructing a nanobody library in mammalian cells. Complementarity-determining region (CDR) random sequences were first incorporated into upstream and downstream dsDNAs by PCR. In the first method, named dsDNA-HR, upstream and downstream dsDNAs containing an identical overlapping sequence were co-transfected into cultured mammalian cells for intracellular homologous recombination that resulted in the formation of an intact nanobody library expression cassette. In the second method, named in vitro ligation, we generated full-length nanobody expression dsDNAs via ligation of restriction digested upstream and downstream dsDNAs. The obtained full-length dsDNAs were transfected into mammalian cells for nanobody library expression. Using both methods, we generated over a million unique nanobody sequences, as revealed by high-throughput sequencing. Single-cell sequencing was employed to resolve the diversity of the dsDNA-HR nanobody library. We also identified a small molecule, Nocodazole, which could enhance the efficacy of dsDNA-HR.
2021-09-21 | GSE158906 | GEO
Project description:Resistance to anti-spike nanobody.
| PRJEB49553 | ENA
Project description:The goal of these experiments was to define the targets of Ty3 transposition in Saccharomyces cerevisiae. Ty3 is a retroviruslike element that is found at the transcription initiation site of chromosomal tRNA genes. A Ty3 that can be induced by growth in galactose-containing medium and which was marked by an insertion of HIS3 downstream of the second open reading frame of the element (POL3) was induced to undergo transposition by plating cells onto galactose containing medium and replica-plating onto medium selective for cells that had undergone transposition. These cells were collected, DNA was extracted, and inverse PCR was performed using primers inside the Ty3 element in order to generate a library of insertion sites flanked by Illumina sequence-compatible primers.
2011-09-02 | E-GEOD-31820 | biostudies-arrayexpress
Project description:Nanobody is one special type of single-domain antibody fragment with multiple advantages over traditional antibody. Our previous work established linear-double-stranded DNA (ldsDNA, or PCR amplicon) as novel biological parts for building AND gate genetic circuits in mammalian cells. During this AND-gate circuit formation process, the co-transfected up- and down-stream ldsDNAs could be linked together to form intact gene expression cassette. Here, we employed this ldsDNA-based AND-gate (LBAG) strategy to construct nanobody library in mammalian cells. The sequence complexity of complementary determining regions (CDRs) was introduced into ldsDNA by PCR amplification. After being co-transfected into mammalian cells, the up- and down- stream ldsDNAs undergo AND gate linkage and form full nanobody coding regions, containing CDR1-3. High throughput sequencing identified 22,173 unique oligonucleotide sequences in total generated by this strategy. Thus, we developed a novel method to construct nanobody library, which is a start point for building high content nanobody library in mammalian cells.
2021-07-24 | GSE155159 | GEO
Project description:Probing epigenomic marks such as histone modifications at a single cell level in thousands of cells has been recently enabled by technologies such as scCUT&Tag. Here we developed a multimodal and optimized iteration of scCUT&Tag called nano-CT (for nano-CUT&Tag) that allows simultaneous probing of three epigenomic modalities at single-cell resolution, using nanobody-Tn5 fusion proteins. nano-CT is compatible with starting materials as low as 25 000 cells and has significantly higher resolution than scCUT&Tag, with a 16-fold increase in the number of fragments per cells. We used nano-CT to simultaneously profile chromatin accessibility, H3K27ac and H3K27me3 in a complex tissue - juvenile mouse brain. The obtained multimodal dataset allowed for discrimination of more cell types/states that scCUT&Tag, and inference of chromatin velocity between ATAC and H3K27ac in the oligodendrocyte (OL) lineage. In addition, we used nano-CT to deconvolute H3K27me3 repressive states and infer two sequential waves of H3K27me3 repression at distinct gene modules during OL lineage progression. Thus, given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in different biological systems at single cell level.
2022-09-20 | GSE198467 | GEO
Project description:Transposable elements (TEs) are genomic parasite that threat genome integrity. One major strategy organisms evolved to balance TE activity in the germline is the Piwi-interacting RNAs (piRNAs) pathway. It prevents transposition by repressing TE at transcriptional and/or post-transcriptional level. However, evidence that TE derepression caused by piRNA pathway impairment is actually followed by bursts of transposition is still lacking. Here we present a genome-wide TE-analyses of the replicative TE life cycle (their expression, their translation to their transposition in the germline) after piRNA pathway impairment in the somatic follicle cells. We observed a high transposition-rate in the germline, leading to a 10-fold increase in genomic TE-load after 70 successive generations of piRNA pathway impairment. Moreover, we demonstrate that siRNAs co-regulate TE activity at post transcriptional level in follicle cells. These observations demonstrate that the small RNA-mediated TE repression in follicle cells contribute to genome homeostasis.
2018-08-09 | GSE112925 | GEO