Patent ID: 11939622
Assignee: BECTON, DICKINSON AND COMPANY
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method comprising:
permeabilizing a cell comprising a nuclear target associated with double-stranded deoxyribonucleic acid (dsDNA), wherein the dsDNA is a genomic DNA (gDNA), and wherein the nuclear target is a protein, wherein the nuclear target comprises a chromatin-associated protein and is attached to the dsDNA;
generating a plurality of nuclear target-associated dsDNA fragments by contacting the nuclear target associated with dsDNA with a digestion composition comprising a DNA digestion enzyme and a binding reagent capable of specifically binding to the nuclear target such that each of plurality of nuclear target-associated dsDNA fragments comprises a single-stranded overhang, wherein the binding reagent comprises a binding reagent specific oligonucleotide comprising a unique identifier sequence for the binding reagent, wherein the DNA digestion enzyme comprises a domain that is capable of specifically binding to the binding reagent, and wherein the DNA digestion enzyme is a restriction enzyme capable of creating a single-stranded restriction site overhang on the dsDNA of the nuclear target associated with dsDNA;
adding a synthetic particle comprising a first plurality of oligonucleotide barcodes and a second plurality of oligonucleotide barcodes to the permeabilized cell after the contacting step;
lysing the permeabilized cell after the adding step;
generating a plurality of barcoded nuclear target-associated DNA fragments by barcoding the plurality of nuclear target-associated dsDNA fragments using the first plurality of oligonucleotide barcodes, wherein each oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a first target-binding region capable of hybridizing to the plurality of nuclear target-associated dsDNA fragments, wherein said barcoding the plurality of nuclear target-associated dsDNA fragments comprises:
hybridizing the first plurality of oligonucleotide barcodes to the plurality of nuclear target-associated dsDNA fragments,
extending the first plurality of oligonucleotide barcodes hybridized to the plurality of nuclear target-associated dsDNA fragments, and/or
ligating the nuclear target-associated dsDNA fragments to the first plurality of oligonucleotide barcodes hybridized to the plurality of nuclear target-associated dsDNA fragments; and
generating a plurality of barcoded binding reagent specific oligonucleotides by barcoding the binding reagent specific oligonucleotide using the second plurality of oligonucleotide barcodes after the lysing step, wherein each oligonucleotide barcode of the second plurality of oligonucleotide barcodes comprises a second target-binding region capable of hybridizing to the binding reagent specific oligonucleotide,
wherein said barcoding the binding reagent specific oligonucleotide comprises hybridizing the second plurality of oligonucleotide barcodes to the binding reagent specific oligonucleotide and extending the second plurality of oligonucleotide barcodes hybridized to the binding reagent specific oligonucleotide, and wherein the binding reagent specific oligonucleotide comprises a sequence complementary to the second target-binding region.