Abstract: A method for detecting the binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in a cell, including: contacting a permeabilized cell or nucleus with a specific binding agent that specifically recognizes the chromatin-associated factor of interest, wherein the specific binding agent is linked to a nuclease that is inactive or an activatable transposome; activating the nuclease or transposase, thereby excising the sequence of chromatin DNA bound to the chromatin-associated factor of interest; isolating the excised DNA; and determining the sequence of the excised DNA, thereby detecting binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in the cell.

Abstract: The disclosure provides methods and related systems and reagents for detecting, sequencing, and/or mapping sites of DNA accessibility in the chromatin of a cell. The method comprises contacting a permeabilized cells with an affinity reagent that specifically binds a nucleosome depleted region (NDR) marker. The first affinity reagent is coupled, directly or indirectly, with at least one transposome. The transposase component of the transposome is activated under low ionic conditions, resulting in cleaving and tagging chromatin DNA. The DNA segment, thus tagged, is excised by virtue of multiple cleavage points, which is then isolated for analysis (e.g., sequencing and mapping). The method can include additional affinity reagents that are similarly functionalized but instead bind to negative regulatory elements in the chromatin, thus allowing for the simultaneous mapping of DNA accessibility and inaccessibility in the genome of a single cell. The methods can be applied to a variety of analytic platforms.

Abstract: A method for detecting the binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in a cell, including: contacting a permeabilized cell or nucleus with a specific binding agent that specifically recognizes the chromatin-associated factor of interest, wherein the specific binding agent is linked to a nuclease that is inactive or an activatable transposome; activating the nuclease or transposase, thereby excising the sequence of chromatin DNA bound to the chromatin-associated factor of interest; isolating the excised DNA; and determining the sequence of the excised DNA, thereby detecting binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in the cell.

Abstract: A method for detecting the binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in a cell, including: contacting a permeabilized cell or nucleus with a specific binding agent that specifically recognizes the chromatin-associated factor of interest, wherein the specific binding agent is linked to a nuclease that is inactive or an activatable transposome; activating the nuclease or transposase, thereby excising the sequence of chromatin DNA bound to the chromatin-associated factor of interest; isolating the excised DNA; and determining the sequence of the excised DNA, thereby detecting binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in the cell.

Abstract: A method for detecting the binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in a cell, including: contacting a permeabilized cell or nucleus with a specific binding agent that specifically recognizes the chromatin-associated factor of interest, wherein the specific binding agent is linked to a nuclease that is inactive or an activatable transposome; activating the nuclease or transposase, thereby excising the sequence of chromatin DNA bound to the chromatin-associated factor of interest; isolating the excised DNA; and determining the sequence of the excised DNA, thereby detecting binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in the cell.

Abstract: The present invention provides compositions, methods, and kits for generating and isolating tagged nuclei of specific cell types with a high yield and purity. The compositions, methods, and kits provided herein comprise expressing in a cell a nuclear envelope tagging fusion polypeptide comprising a nuclear envelope targeting domain and an affinity reagent binding region. In some embodiments, expression of the fusion polypeptide is under the control of a cell type-specific promoter. Some embodiments also comprise expressing in a cell a biotin ligase, wherein the affinity reagent binding region comprises a biotin ligase accepting site, and wherein at least one of the nuclear envelope tagging fusion polypeptide and the biotin ligase is expressed under the control of a cell type-specific promoter.

Abstract: The present invention provides a simple and general reverse genetic strategy for use in organisms where well-developed genetic tools are lacking. In particular, the method comprises contacting an organism or cell with a mutagen which primarily induces point mutations in the genomic DNA; preparing DNA from the organism or cell; amplification of a genomic region of interest; and screening for changes in the mutagenized DNA sequence in the genomic region of interest as compared to the parent organism or cell. Screening for mutations can comprise detection of heteroduplexes to identify a mutant organism or cell. The methods of the present invention are particularly useful for identifying functional mutations in plants.

Abstract: A novel technique is provided, designated DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering a DNA modification enzyme, in particular, E. coli DNA adenine methyl transferase (Dam), to a chromatin protein. The DNA modification enzyme (Dam) can be targeted in vivo to the native binding loci of the protein, resulting in local DNA modification. Sites of DNA modification can subsequently be mapped using modification-specific restriction enzymes, antibodies, or DNA array methods. DNA Modification Identification (DamID) has potential for genome-wide mapping of in vivo target binding sites of chromatin proteins in various eukaryotes.

Abstract: A process is provided for producing an ordered series of cloned, circular, DNA molecules containing shortened target DNA segments derived from a long target DNA segment, which are suitable for use in determining the nucleotide sequence of the long target DNA segment, or for targeting specific regions within the target DNA segment. The process includes producing, by molecular cloning, a plurality of double-stranded recombinant DNA molecules each containing: (i) vector DNA; (ii) a sequencing primer binding site; and, (iii) a DNA region having unique endonuclease sites and a long target DNA segment. The sequencing primer binding site is spaced from the long target DNA segment by at least a portion of said DNA region having the unique endonuclease sites. The plurality of double-stranded circular recombinant DNA molecules are cleared using two restriction endonucleases.

Abstract: A process is provided for producing an ordered series of cloned, circular, DNA molecules containing shortened target DNA segments derived from a long target DNA segment, which are suitable for use in determining the nucleotide sequence of the long target DNA segment, or for targeting specific regions within the target DNA segment. The process includes producing, by molecular cloning, a plurality of double-stranded recombinant DNA molecules each containing: (i) vector DNA; (ii) a sequencing primer binding site; and, (iii) a DNA region having unique endonuclease sites and a long target DNA segment. The sequencing primer binding site is spaced from the long target DNA segment by at least a portion of said DNA region having the unique endonuclease sites. The plurality of double-stranded circular recombinant DNA molecules are cleared using two restriction endonucleases.