Granted Patent B2

US 12,709,767 · App. 18/353,058 · Granted Aug 18, 2026

Inventors: David A. Weitz (Cambridge, MA); Huidan Zhang (Cambridge, MA); John Heyman (Cambridge, MA); Allon Moshe Klein (Cambridge, MA)

Assignee: President and Fellows of Harvard College

C12P19/34C12N15/1006C12N15/1065C12N15/1093C12Q1/6869C12Q2600/156

Abstract

Claims (21)

1 . A method, comprising:

(a) providing a plurality of droplets comprising particles such that at least about 90% of the droplets contains one particle or no particle, the particles comprising oligonucleotides, the oligonucleotides comprising an adapter sequence that is identical for all of the plurality of droplets and particles, and a first barcode selected from a pre-defined pool of first barcodes, and a second barcode selected from a pre-defined pool of second barcodes, such that substantially each of the particles comprises distinguishable barcode sequences;

(b) encapsulating cells within the plurality of droplets at a density of less than 1 cell/droplet;

(c) lysing at least some of the cells within the plurality of droplets to release nucleic acid from the cells;

(d) attaching a released nucleic acid sequence to the oligonucleotides via covalent bonding after lysing at least some of the cells; and

(e) selectively amplifying the attached released nucleic acid sequence by:

(i) exposing the adapter sequence to a gene-specific inner forward primer comprising a sequence complementary to the adapter sequence and a sequence complementary to the released nucleic acid sequence; and

(ii) exposing the gene-specific inner forward primer and a gene-specific reverse primer to the released nucleic acid sequence, wherein the concentration of the gene-specific inner forward primer is lower than the concentration of the gene-specific reverse primer;

thereby generating an amplicon, wherein the amplicon is further amplified using the oligonucleotide and the gene-specific reverse primer to produce an oligonucleotide comprising the first barcode, the second barcode, and the amplicon.

2 . The method according to claim 1 , wherein covalent bonding occurs by enzymatic attachment or ligation.

3 . The method according to claim 1 , wherein, prior to (d), the oligonucleotides and the released nucleic acid sequence both have sticky ends.

4 . The method according to claim 3 , wherein the released nucleic acid sequence comprises an overhang region and the oligonucleotides comprise a complementary overhang region, and wherein the overhang region and the complementary overhang region contain a single nucleotide, or a random sequence of nucleotides, or a restriction site.

5 . The method according to claim 1 , wherein the pre-defined pool of first barcodes comprises at least about 300 distinguishable barcodes.

6 . The method according to claim 1 , wherein the pre-defined pool of second barcodes comprises at least about 300 distinguishable barcodes.

7 . The method according to claim 1 , wherein the distinguishable barcode sequences comprise at least 10,000 distinguishable barcode sequences.

8 . The method according to claim 1 , wherein at least some of the oligonucleotides are attached to the surface of the particles.

9 . The method according to claim 1 , wherein at least some of the oligonucleotides are covalently bonded to the particles.

10 . The method according to claim 1 , wherein at least some of the oligonucleotides comprise a cleavable linker.

11 . The method according to claim 1 , further comprising releasing at least some of the oligonucleotides from the particles prior to lysing at least some of the cells.

12 . The method according to claim 1 , wherein the particles are contained within the droplets at no more than about 1 particle/droplet.

13 . The method according to claim 1 , wherein the plurality of droplets further comprise DNA-tagged antibodies.

Assignments (1)

Continuity (3)

References Cited (362)