IP Library › Granted Patent US 12,545,908
Granted Patent B2
US 12,545,908 · App. 15/971,417 · Granted Feb 10, 2026

Methods for trapping and barcoding discrete biological units in hydrogel

Inventor: Stuart Edelstein (Paris, FR)
Assignee: SCIPIO BIOSCIENCE
C12N15/1096A61K47/06A61K47/6903C12Q1/682C12Q1/6823C12Q1/6869C12Q1/6876C12Q1/6809C12Q2563/179C12Q2563/185
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Quick Facts
Patent No.
US 12,545,908
App. No.
15/971,417
Granted
Feb 10, 2026
Kind
B2
Abstract

Disclosed are methods for trapping and barcoding discrete biological units in a hydrogel. Also disclosed are methods for analyzing gene expression, genotype, haplotype or epigenome in discrete biological units, as well as kits for implementing the methods of the present disclosure.

Claims (46)

1 . A method for trapping thousands of biological units in a single hydrogel and barcoding their nucleic acids, said method comprising the steps of:

a) binding thousands of biological units on thousands of barcode beads, wherein each barcode bead comprises a unique barcode present in multiple clonal copies, and wherein the barcode beads comprise at least one means involved with binding biological units and/or the biological units comprise at least one means involved with binding barcode beads, to form thousands of biological unit/unique barcode bead complexes;

b) contacting said thousands of complexes with a hydrogel solution;

c) polymerizing the hydrogel solution to embed, discretely from one another, said thousands of complexes in a single hydrogel matrix formed from the hydrogel solution;

d) releasing nucleic acids from the biological unit of each biological unit/unique barcode bead complex present in the hydrogel matrix, wherein the hydrogel matrix has a pore size preventing the diffusion of said nucleic acids;

e) for each biological unit/unique barcode bead complex, annealing the unique barcode of the barcode bead of each biological unit/unique barcode bead complex to the nucleic acids of the biological unit of the same biological unit/unique barcode bead complex;

f) depolymerizing the hydrogel matrix; and

g) in-bulk synthetizing a nucleic acid library from the nucleic acids by primer-directed extension of the barcode,

wherein the method does not utilize polydimethylsiloxane (“PDMS”) chips or droplets to embed the complexes.

2 . The method according to claim 1 , wherein said method is for analyzing gene expression in thousands of cells, and said method comprises the steps of:

a) binding thousands of cells with thousands of barcode beads, to form thousands of cell/unique barcode bead complexes,

b) contacting said thousands of complexes with a hydrogel solution,

c) polymerizing the hydrogel solution to embed, discretely from one another, said thousands of complexes in a single hydrogel matrix formed from the hydrogel solution,

d) releasing messenger RNAs from the cells of each cell/unique barcode bead complex present in the hydrogel matrix,

e) for each cell/unique barcode bead complex, annealing the unique barcode of the barcode bead of each cell/unique barcode bead complex to the messenger RNAs of the cell of the same cell/unique barcode bead complex,

f) depolymerizing the hydrogel matrix,

g) in-bulk synthetizing a barcoded cDNA library from the messenger RNAs by primer-directed extension of the barcode, and

h) sequencing the barcoded cDNA library.

3 . The method according to claim 1 , wherein said method is for analyzing the genotype or haplotype in thousands of cells, and said method comprises the steps of:

a) binding thousands of cells with thousands of barcode beads, wherein each barcode unit comprises clonal copies of a unique barcode, to form thousands of cell/unique barcode bead complexes,

b) contacting said thousands of complexes with a hydrogel solution,

c) polymerizing the hydrogel solution to embed, discretely from one another, said thousands of complexes in a single hydrogel matrix formed from the hydrogel solution,

d) releasing genomic DNA from each cell present in the hydrogel matrix,

e) for each cell/unique barcode bead complex, annealing the unique barcode of the barcode bead with the genomic DNA,

f) depolymerizing the hydrogel matrix,

g) in-bulk synthetizing a barcoded DNA library from the genomic DNA by primer-directed extension of the barcode, and

h) sequencing the barcoded DNA library.

4 . The method according to claim 1 , wherein said method is for analyzing the epigenome in thousands of cells, and said method comprises the steps of:

a) binding thousands of cells with thousands of barcode beads, wherein each barcode unit comprises clonal copies of a unique barcode, and

wherein the barcode beads comprise at least one means involved with binding cells and/or the cells comprise at least one means involved with binding barcode beads, to form thousands of cell/unique barcode bead complexes,

b) contacting said thousands of complexes with a hydrogel solution,

c) polymerizing the hydrogel solution to embed, discretely from one another, said thousands of complexes in a single hydrogel matrix formed from the hydrogel solution,

d) releasing non-nucleosome-bound-DNA from the cells of each cell/unique barcode bead complex present in the hydrogel matrix,

e) for each cell/unique barcode bead complex, annealing the unique barcode of the barcode bead with the non-nucleosome-bound-DNA,

f) depolymerizing the hydrogel matrix,

g) in-bulk synthetizing a barcoded non-nucleosome-bound-DNA library from the non-nucleosome-bound-DNA by primer-directed extension of the barcode, and

h) sequencing the barcoded non-nucleosome-bound-DNA library.

5 . The method according to claim 1 , wherein said biological units are immobilized on a support.

6 . The method according to claim 1 , wherein said barcode beads are immobilized on a support.

7 . The method according to claim 1 , wherein said unique barcode comprises a nucleic acid sequence barcode.

8 . The method according to claim 1 , wherein said unique barcode further comprises a nucleic acid sequence primer.

9 . The method according to claim 8 , wherein said nucleic acid sequence primer comprises random nucleic acid sequence primers and/or specific nucleic acid sequence primers.

10 . The method according to claim 1 , wherein said at least a means involved with binding said biological unit comprises proteins, peptides and/or fragments thereof; antibodies and/or fragments thereof; nucleic acids; carbohydrates; vitamins and/or derivatives thereof; coenzymes and/or derivatives thereof; receptor ligands and/or derivatives thereof, and/or hydrophobic groups.

11 . The method according to claim 1 , wherein said discrete biological units comprise cells, groups of cells, viruses, nuclei, mitochondria, chloroplasts, biological macromolecules, exosomes, chromosomes, contiguity preserved transposition DNA fragments and/or nucleic acid fragments.

12 . The method according to claim 11 , wherein said cells or groups of cells comprise cells in in vitro culture, stem cells, tumor cells, tissue biopsy cells, blood cells and tissue section cells.

13 . The method according to claim 9 , wherein the specific nucleic acid sequence primers comprise a poly-dT or poly-dU sequence.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 14, 2018
From: EDELSTEIN, STUART
To: SCIPIO BIOSCIENCE
Reel/Frame 046087/0438 →
Continuity (2)
Provisional Application 62502180 · May 5, 2017
Related Publication 20180320173A1 · Nov 8, 2018
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