Methods for barcoding macromolecules in individual cells

Patent metadata

Field	Value
Publication number	US-12428673-B2
Application number	US-202318322490-A
Country	US
Kind code	B2
Filing date	May 23, 2023
Priority date	Nov 10, 2021
Publication date	Sep 30, 2025
Grant date	Sep 30, 2025

Abstract

The present disclosure relates to methods and kits for generating single cell barcodes and imparting them to the constituent molecules within a single cell. Additionally, methods to overlay sample barcode and spatial barcode information onto the single cell barcodes are also described. Generation of single cell barcodes is achieved by labeling the genomic DNA of a cell/nucleus with a small handful, preferably just a one or two cellular barcode probes (CBP) that can be amplified and propagated to label the constituent molecules within the cell. The disclosure finds utility in applications such as characterization of cellular heterogeneity, comprehensive profiling of tissue composition, characterization of adherent cells, discovery of new cell subtypes and functions of individual cells in the context of its microenvironment, and others.

First claim

What is claimed is:

1. A method for barcoding macromolecules from a sample comprising a population of cells, the method comprising the following steps:
a) permeabilizing cells, and/or nuclei of the cells, from the population of cells of the sample;
b) optionally making genomic DNA of the permeabilized cells and/or nuclei at least partially accessible to nucleic acid hybridization;
c) delivering cell barcode probes to the permeabilized cells and/or nuclei of the permeabilized cells, wherein a given cell barcode probe comprises a genome binding element shared among the cell barcode probes, and a cell barcode unique for the given cell barcode probe, and wherein the genome binding element hybridizes to a region in the genomic DNA, thereby forming a nucleic acid duplex between the genome binding element and the region of the genomic DNA in the cells and/or nuclei;
d) removing cell barcode probes that are not bound to the genomic DNA from the cells and/or nuclei, whereby no more than a defined number of copies of the cell barcode probe remain in each cell or nucleus;
e) partitioning the cells and/or nuclei into a plurality of compartments;
f) amplifying the cell barcodes within compartments of the plurality of compartments, thereby forming amplified cell barcodes within the compartments; and
g) attaching the amplified cell barcodes to the macromolecules within the compartments, thereby forming barcoded macromolecules.

2. The method of claim 1 , further comprising releasing the barcoded macromolecules from the compartments.

3. The method of claim 1 , wherein the macromolecules being barcoded are polypeptides, mRNA molecules or cDNA molecules.

4. The method of claim 1 , wherein the region in the genomic DNA is a non-repetitive region, optionally wherein the non-repetitive region in the genomic DNA is a non-coding region or a differentially methylated region.

5. The method of claim 1 , wherein the defined number of copies is one copy or two copies.

6. The method of claim 1 , wherein the sample is a spatial sample, and wherein the sample is dissociated into a plurality of cells at step (e).

7. The method of claim 6 , wherein each of the cell barcode probes further comprise a positional barcode different for at least some of the cell barcode probes.

8. The method of claim 6 , wherein the cell barcode probes are delivered at step (c) from a spatially ordered array.

9. The method of claim 6 , further comprising, after step (b),
(i) delivering a plurality of positional probes to the permeabilized cells and/or nuclei, wherein a given positional probe comprises a common targeting element configured to be attached to the macromolecules and a positional barcode different for each positional probes; and
(ii) attaching positional probes from the plurality of positional probes to the macromolecules.

10. The method of claim 9 , wherein each of the amplified cell barcodes comprises a common region that is configured to hybridize to a region in the positional probes; and the method further comprises a step of performing a primer extension reaction to transfer the amplified cell barcodes to the positional probes attached to the macromolecules.

11. The method of claim 9 , wherein the plurality of positional probes is delivered from a spatially ordered array.

12. The method of claim 1 , wherein each compartment of the plurality of compartments comprises a compartment barcode configured to be attached to the macromolecules.

13. The method of claim 1 , wherein during partitioning the cells and/or nuclei into the plurality of compartments at step (e), on average no more than one cell or nucleus comprising a cell barcode probe is comprised within a single compartment.

Assignees

• Encodia Inc

Inventors

• Chee Mark S
• Gong Haibiao
• Gunderson Kevin L

Classifications

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q1/6806

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