Chromatin immunocapture devices and methods of use

Patent metadata

Field	Value
Publication number	US-10597698-B2
Application number	US-201515121667-A
Country	US
Kind code	B2
Filing date	Mar 3, 2015
Priority date	Mar 3, 2014
Publication date	Mar 24, 2020
Grant date	Mar 24, 2020

Abstract

This application provides fluidic devices, such as microfluidic devices, which can be used for the creation and/or manipulation of droplets in droplet-based microfluidic systems, as well as systems and methods for using the same. The microfluidic devices can be used to generate droplets, extract or inject volume to droplets, and/or split droplets. Also provided are methods for generating nucleosomes, and isolated DNA from nucleosomes (or from non-nucleosomes), for example using the disclosed devices.

First claim

We claim:

1. A method of analyzing non-nucleosomal DNA, comprising:
encapsulating an aqueous solution comprising a single cell and a lysing solution within a single droplet, wherein the single cell comprises nucleosomes, and wherein the single droplet comprises oil and a surfactant, thereby lysing the cell within the single droplet;
adding to the single droplet a nuclease solution, thereby cleaving DNA in the single droplet;
adding to the single droplet a quenching solution, thereby quenching the nuclease;
adding to the single droplet a solution containing a first solid support comprising one or more binding reagents that specifically bind to all nucleosomes;
allowing the first solid support to bind to its target within the single droplet;
separating the solid support from non-nucleosomal DNA in the single droplet;
adding to the single droplet a second solid support coupled to a reagent that binds the non-nucleosomal DNA;
separating the single droplet into a portion containing the second solid support and a portion containing the non-nucleosomal DNA;
collecting the portion of the single droplet containing the non-nucleosomal DNA; and
analyzing the non-nucleosomal DNA.

2. The method of claim 1 , wherein collecting the portion of the single droplet containing the non-nucleosomal DNA comprises combining the non-nucleosomal DNA from a plurality of single droplets.

3. A method of analyzing DNA associated with nucleosomes, comprising:
encapsulating an aqueous solution comprising a single cell and a lysing solution within a single droplet, wherein the single cell comprises nucleosomes, and wherein the single droplet comprises oil and a surfactant, thereby lysing the cell within the single droplet;
adding to the single droplet a non-nucleosome-cleaving nuclease solution, thereby cleaving DNA in the single droplet without substantially affecting portions of the DNA that interact with histones within the nucleosomes;
adding to the single droplet a quenching solution, thereby quenching the non-nucleosome-cleaving nuclease;
adding to the single droplet a solution containing a first solid support comprising one or more binding reagents that specifically bind to a histone, histone variant, histone mark, histone regulator, transcription factor or modified bases within DNA;
allowing the first solid support to bind to its target within the single droplet;
concentrating the first solid support bound to its target and the DNA;
adding to the single droplet an enzyme solution that releases DNA from the solid support;
adding to the single droplet a second solid support coupled to a reagent that binds the released DNA;
separating the single droplet into a portion containing the second solid support and a portion containing the DNA;
collecting the portion of the single droplet containing the DNA; and
analyzing the DNA.

4. The method of claim 1 , wherein analyzing the non-nucleosomal DNA comprises amplification of the non-nucleosomal DNA and/or sequencing the non-nucleosomal DNA.

5. The method of claim 4 , wherein amplification of the non-nucleosomal DNA comprises digital droplet polymerase chain reaction (ddPCR).

6. The method of claim 1 , wherein the adding steps comprise adding reagents to the single droplet by destabilizing the single droplet with a high voltage (HV) field.

7. The method of claim 1 , wherein the single cell within a single droplet is present in a curved microchannel.

8. The method of claim 1 , wherein a plurality of single cells within a plurality of single droplets are analyzed contemporaneously.

9. The method of claim 1 , wherein the first solid support comprises magnetic beads.

10. The method of claim 1 , wherein one or more of the steps are automated.

11. The method of claim 1 , wherein DNA and protein complexes within the cell are crosslinked prior to encapsulating the cell within the single droplet.

12. The method of claim 1 , wherein the single cell is a eukaryotic cell.

13. The method of claim 12 , wherein the eukaryotic cell is a human cell, invertebrate cell, amphibian cell, or bird cell.

Assignees

• Univ Illinois
• Mayo Found Medical Education & Res

Inventors

• Bailey Ryan
• Tice Joshua
• Ordog Tamas
• Lee Jeong Heon
• Graybill Richard Martin
• Xu Yi
• Doonan Steven

Classifications

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• B01L2200/0673

  Handling of plugs of fluid surrounded by immiscible fluid · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01L2300/0867

  Multiple inlets and one sample wells, e.g. mixing, dilution · CPC title