Single-Cell Epigenomic Profiling Using Droplet Fluidics and Hydrogels
AssigneesCZ Biohub SF, LLC, The Regents of the University of California
InventorsAdam Abate, Xiangpeng Li
Life of this patent
Estimating the term…
Abstract
The present disclosure provides materials and methods for partitioning cells and high throughput, single-cell epigenetic sequencing. Methods for using chemically-modified hydrogels are also provided herein. Wherein determining the epigenomic state of a single-cell comprises preparing a functionalized hydrogel, wherein said functionalized hydrogel is chemically modified with a functional group capable of binding to a capture reagent.
Claims10
A method of determining the epigenomic state of a single cell comprising the steps of:
- (a) preparing a functionalized hydrogel, wherein said functionalized hydrogel is chemically modified with a functional group capable of binding to a capture reagent;
- (b) preparing a functionalized capture reagent, wherein said functionalized capture reagent is chemically modified with a functional group capable of binding to the functional group of the functionalized hydrogel of (a), and wherein said capture reagent is also capable of binding to a nucleic acid;
- (c) encapsulating single cells into particles comprising a cell lysis buffer and the functionalized hydrogel of (a) under conditions that allow cell lysis;
- (d) preparing a nucleic acid from the encapsulated single cells of (c) under conditions that allow binding of the capture reagent to (i) the nucleic acid, and (ii) the functional group of the functionalized hydrogel, thereby forming a captured nucleic;
- (e) preparing the captured nucleic acid of (d) for sequencing, wherein said preparing comprises universal adaptor ligation and barcoding;
- and
- (f) sequencing the captured nucleic acid and thereby determining the epigenomic state of a single cell.
The method of any of the preceding claims, wherein the functional group capable of binding to the functional group of the functionalized hydrogel is selected from the group consisting of azide (N3), dibenzocyclooctyne (DBCO), alkyne, tetrazine (TZ), methyltetrazine, trans-cyclooctene (TCO), cyclooctene, norbornene (NZ), cyclopropene, thiol, bromo, tosylate, maleimide, amine, carboxylic acid and NHS ester.
The method of any of the preceding claims, wherein the capture reagent is selected from the group consisting of an antibody, streptavidin, avidin, and aptamers.
The method of any of the preceding claims, wherein the nucleic acid is genomic DNA.
The method of any of the preceding claims, wherein the conditions of (d) that allow binding of the capture reagent to (i) the nucleic acid, and (ii) the functional group of the functionalized hydrogel comprises cross-linking the capture reagent to the hydrogel.
Dependencies are inferred from the claim text.