# US 10,479,986 B2 · Nucleic acid constructs and methods for labeling and detecting nucleosomal DNA modifications

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- Number: US 10,479,986 B2
- Document ID: US10479986B2
- Status: Granted
- Kind: B2
- Application number: 15/607,244
- Filed: 2017-05-26
- Published: 2017-11-30
- Granted: 2019-11-19
- Earliest claimed: 2016-05-27
- Assignees: The Broad Institute, Inc.; The General Hospital Corporation
- Applicants: The Broad Institute, Inc.; The General Hospital Corporation
- Inventors: Charles B. Epstein; Peter Van Galen; Bradley E. Bernstein
- Web page: https://patentgrep.com/patent/US10479986B2

## CPC

- C12N 9/64: derived from animal tissue
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- G01N 33/00: Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Embodiments disclosed herein provide methods for identifying cell-type-specific nucleosomal DNA modifications. The methods leverage nucleosomal DNA barcoding and pool-and-split multiplexing to provide high-throughout, quantitative profiling of nucleosomal DNA states. The methods enable the profiling of multiple nucleosomal DNA marks across different cell types and/or conditions thereby linking quantitative changes in chromatin landscapes to different genotypes and chemical and physical perturbations.

## Claims

### Claim 1

A method for labeling nucleic acids from a cell or population of cells comprising:
providing one or more individual discrete volumes, each individual discrete volume comprising a cell or population of cells;
lysing the cell or population of cells in each individual discrete volume and fragmenting nucleosomal DNA from the lysed cell or population of cells;
labeling the fragmented nucleosomal DNA in each individual discrete volume with a barcoded adapter on at least one free end of the fragmented nucleosomal DNA, wherein:
the barcoded adapter comprises an amplification promoter adjacent to a first or second read sequencing primer binding site which is adjacent to a barcode sequence,
the labeled nucleosomal DNA comprises, in a 3′ to 5′ orientation, a first read sequencing primer binding site, the nucleosomal DNA sequence, the barcode sequence, and a second read sequencing primer binding site,
the barcode sequence is unique to each individual discrete volume thereby identifying a sample from which the labeled nucleosomal DNA originated;
isolating and sequencing the labeled nucleosomal DNA from each sample; and
grouping the sequenced nucleosomal DNA by common barcode thereby identifying the individual discrete volumes from which the nucleosomal DNA originated.

### Claim 2 (depends on 1)

The method of claim 1, wherein sequencing the labeled nucleosomal DNA comprises:
generating amplified RNA copies of the labeled nucleosomal DNA by in vitro transcription from the amplification promoter on the barcoded adapter;
generating cDNA from the amplified RNA copies using a reverse transcription primer comprising a random hexamer and a second sequencing primer binding site; and
generating a dsDNA copy of the cDNA for sequencing by amplification using an amplification primer pair, and each primer in an amplification primer pair comprising a sequencing primer binding site and sequencing adapter, wherein the resulting dsDNA copy incorporates the first and second read sequencing primer binding sites and a sequencing adapter on both ends of the dsDNA copy.

### Claim 3 (depends on 2)

The method of claim 2, wherein the primer pair used on each sample incorporates a second barcode during amplification that identifies the nucleosomal modification detected by the nucleosomal modification detection assay performed on that assay sample.

### Claim 4 (depends on 2)

The method of claim 2, wherein the first sequencing primer binding site of the adapter is a first read sequencing primer binding site, and the second sequencing primer binding site on the reverse transcription primer is a second read sequencing primer binding site.

### Claim 5 (depends on 1)

The method of claim 1, further comprising conducting one or more nucleosomal modification detection assays on each individual discrete volume, wherein grouping the sequenced nucleosomal DNA by common barcode further identifies epigenetic modifications in the cell or population of cells in each individual discrete volume.

### Claim 6 (depends on 5)

The method of claim 5, wherein the one or more nucleosomal modification detection assay is a chromatin immunoprecipitation assay.

### Claim 7 (depends on 6)

The method of claim 6, wherein the chromatin immunoprecipitation assay detects one or more chemical modifications of histones.

### Claim 8 (depends on 5)

The method of claim 5, further comprising pooling all labeled nucleosomal DNA and then splitting the nucleosomal DNA into separate assay samples for each nucleosomal modification detection assay to be conducted.

### Claim 9 (depends on 1)

The method of claim 1, wherein the barcoded adapter is double-stranded.

### Claim 10 (depends on 1)

The method of claim 1, wherein digestion of the nucleosomal DNA is done using a micrococcal nuclease (MNase).

### Claim 11 (depends on 1)

The method of claim 1, wherein the individual discrete volume is an individual reaction tube or well of a microwell plate.

### Claim 12 (depends on 1)

The method of claim 1, wherein the individual discrete volume is a droplet generated on a microfluidic device.

### Claim 13 (depends on 1)

The method of claim 1, wherein labeling the fragmented nucleosomal DNA in each individual discrete volume with barcoded adapters comprises delivering to each individual discrete volume a unique set of barcoded adapters, wherein all barcoded adapters in the set identify the individual discrete volume and each individual barcoded adapter in a given set comprises a unique barcode sequence.

### Claim 14 (depends on 13)

The method of claim 13, wherein the set comprises four adapters wherein each individual adapter in the set comprises a unique barcode sequence such that between the four adapters the nucleosides adenine, guanine, cytosine, or thymine are represented at each position in one of the adapter barcode sequences.