# US 12,215,379 B2 · In situ analysis of chromatin interaction

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,215,379 B2
- Document ID: US12215379B2
- Status: Granted
- Kind: B2
- Application number: 17/177,501
- Filed: 2021-02-17
- Published: 2021-08-19
- Granted: 2025-02-04
- Earliest claimed: 2020-02-17
- Assignees: 10x Genomics, Inc.
- Applicants: 10x Genomics, Inc.
- Inventors: Patrik Ståhl; Pelin Sahlén
- Web page: https://patentgrep.com/patent/US12215379B2

## CPC

- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/6841: In situ hybridisation
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

In some embodiments described herein are methods performed in situ for analyzing chromatin interaction events in a cell or in cells of a sample such as a non-homogenized tissue sample. The methods can comprise the spatial analysis of chromatin interaction events across cell populations in a biological sample. The methods can further comprise obtaining a biological sample, hybridizing probes to target nucleic acid sequences involved in chromatin interaction events and producing a nucleic acid sequence comprising all or part of the target nucleic acid sequences, amplifying the nucleic acid sequence so produced and detecting the amplified nucleic acid sequence in situ.

## Claims

### Claim 1

A method, comprising:
(a) contacting a sample with a first chromatin accessing probe and a second chromatin accessing probe simultaneously or sequentially in either order, wherein:
the sample comprises a first chromatin region and a second chromatin region, wherein the first chromatin region and the second chromatin region are separated by a genomic distance of at least 0.5 kilobases (kb), and wherein the first and second chromatin regions interact with each other in a cell and are in the same chromosome in the cell, and
the first chromatin accessing probe hybridizes to the complementary strand of a first nucleic acid strand in the first chromatin region and the second chromatin accessing probe hybridizes to the complementary strand of a second nucleic acid strand in the second chromatin region,
thereby allowing access for binding to the first and second nucleic acid strands;
(b) contacting the sample with a first detection probe and a second detection probe simultaneously or sequentially in either order, wherein:
the first detection probe hybridizes to the first nucleic acid strand and the second detection probe hybridizes to the second nucleic acid strand;
(c) contacting the sample with a first bridging probe that bridges the 3′ end of the first detection probe and the 5′ end of the second detection probe, and a second bridging probe that bridges the 3′ end of the second detection probe and the 5′ end of the first detection probe;
(d) circularizing the first and second bridging probes using the first and second detection probes as templates to form a circular probe, wherein the circular probe comprises a sequence of the first nucleic acid strand and a sequence of the second nucleic acid strand;
(e) generating a rolling circle amplification (RCA) product of the circular probe in situ in the sample; and
(f) detecting the RCA product.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first and second chromatin accessing probes are peptide nucleic acid (PNA) probes or locked nucleic acid (LNA) probes.

### Claim 3 (depends on 2)

The method of claim 2, wherein the melting temperature (Tm) of the hybridization between the first or second nucleic acid strand and the corresponding complementary strand is lower than the Tm of the hybridization between the first or second chromatin accessing probe and the corresponding complementary strand.

### Claim 4 (depends on 1)

The method of claim 1, wherein the interaction between the first and second chromatin regions in the cell is mediated by one or more chromatin associated factors.

### Claim 5 (depends on 1)

The method of claim 1, further comprising treating the sample with a cross-linking agent prior to the contacting step, wherein a nucleic acid in the first and/or second chromatin regions is cross-linked to one another and/or to one or more chromatin associated factors by the cross-linking agent.

### Claim 6 (depends on 1)

The method of claim 1, wherein the sample is contacted with the first and/or second chromatin accessing probes at a probe concentration of at least 100 nM, 200 nM, 500 nM, 1 μM, 2 μM, or more than 2 μM.

### Claim 7 (depends on 1)

The method of claim 1, wherein the sample is contacted with the first and/or second chromatin accessing probes at a temperature of at least 40° C., 45° C., 50° C., or more than 50° C.

### Claim 8 (depends on 1)

The method of claim 1, wherein the sample is incubated with the first and/or second chromatin accessing probes for at least 30 minutes, 1 hours, 2 hours, 5 hours, or more than 5 hours.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first chromatin accessing probe comprises one or more barcode sequences corresponding to a nucleic acid sequence of interest in the first chromatin region.

### Claim 10 (depends on 1)

The method of claim 1, wherein the second chromatin accessing probe comprises one or more barcode sequences corresponding to a nucleic acid sequence of interest in the second chromatin region.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first detection probe comprises one or more barcode sequences corresponding to a nucleic acid sequence of interest in the first chromatin region.

### Claim 12 (depends on 1)

The method of claim 1, wherein the second detection probe comprises one or more barcode sequences corresponding to a nucleic acid sequence of interest in the second chromatin region.

### Claim 13 (depends on 1)

The method of claim 1, wherein the circular probe comprises one or more barcode sequences corresponding to a nucleic acid sequence of interest in the first chromatin region and/or one or more barcode sequences corresponding to a nucleic acid sequence of interest in the second chromatin region.

### Claim 14 (depends on 1)

The method of claim 1, wherein the RCA product is detected in situ in the sample.

### Claim 15 (depends on 1)

The method of claim 1, wherein the detecting comprises imaging the sample using fluorescent microscopy.

### Claim 16 (depends on 1)

The method of claim 1, wherein the genomic distance between the first and second chromatin regions is at least 5 kb.

### Claim 17 (depends on 1)

The method of claim 1, wherein the genomic distance between the first and second chromatin regions is at least 50 kb.

### Claim 18 (depends on 1)

The method of claim 1, wherein the first and second chromatin regions comprise a promoter, an enhancer, a silencer, an insulator, and/or a locus control region (LCR).

### Claim 19 (depends on 1)

The method of claim 1, wherein the interaction between the first and second chromatin regions in the cell is mediated by a transcription factor, an activator, a repressor, a chromatin-remodeler, a polymerase, a replication factor, a DNA repair factor, a histone, a histone-modifying enzyme, and/or a DNA-modifying enzyme.

### Claim 20 (depends on 1)

The method of claim 1, wherein the genomic distance between the first and second chromatin regions is at least 1 kb.