Granted Patent B2

US 12,680,138 · App. 17/825,437 · Granted Jul 14, 2026

Inventor: Robert Meltzer (Belmont, MA)

Assignee: Illumina, Inc.

C12Q1/6886C12N15/1065C12N15/1096C12Q1/6841C12Q2600/156C12Q2600/158

Abstract

Claims (32)

1 . A method for gene expression profiling, the method comprising:

incubating a tissue sample on an array comprising a plurality of labels having binding specificity for a cellular protein and having a nucleic acid index identifying the location of the label on the array, wherein the labels are lyophilized and released upon wetting of the array;

disaggregating a plurality of cells from the tissue sample;

combining the disaggregated plurality of cells with template particles; and

generating a plurality of uniform partitions substantially instantly that encapsulate a single one of the template particles and a single one of the disaggregated cells to form pre-templated instant partitions (PIPs); and

releasing in each PIP nucleic acid molecules and proteins from the disaggregated cells, including proteins bound by a label from the array.

2 . The method of claim 1 , wherein the labels comprise a DNA-conjugated antibody or fragment, a DNA-conjugated aptamer, or a DNA-conjugated lipid.

3 . The method of claim 1 , wherein the step of releasing nucleic acid molecules from the cells comprises releasing RNA molecules.

4 . The method of claim 3 , wherein each of the nucleic acid molecules and labels released from a cell in each PIP is provided a nucleic acid barcode unique to the PIP.

5 . The method of claim 4 , wherein each of the nucleic acid molecules is provided with a unique molecular barcode.

6 . The method of claim 5 , wherein the step of providing a nucleic acid barcode unique to the PIP and a unique molecular identifier comprises the step of reverse transcribing RNA molecules to form cDNA complements comprising the unique molecular identifier and barcode unique to the PIP.

7 . The method of claim 6 , further comprising the step of sequencing the cDNA complements to generate sequence reads associated with each RNA molecule.

8 . The method of claim 7 , further comprising the step of associating each label from a PIP with each sequence read associated with an RNA molecule from the same PIP to generate a set of sequence reads for each label.

9 . The method of claim 1 , wherein the step of incubating the tissue sample comprises:

i) permeabilizing the tissue sample;

ii) staining the tissue sample; and/or

iii) imaging the tissue sample.

10 . The method of claim 1 , wherein the labels have specificity for a nuclear envelope cell protein, disaggregated proteins, or nuclear proteins.

11 . The method of claim 1 , wherein each index identifies at least the X and/or Y coordinates of the label on the array.

12 . The method of claim 11 , wherein each location on the array comprises at least two labels together identifying the X and Y coordinates of the location on the array.

13 . The method of claim 1 , wherein the array is a glass array.

14 . The method of claim 13 , wherein the labels are spotted onto the glass array.

15 . The method of claim 13 , wherein the labels are covalently bonded to the glass array.

16 . The method of claim 14 , wherein the labels are covalently bonded to the glass array by siloxane chemistry.

17 . The method of claim 16 , wherein the labels are covalently bonded to the glass array by their index.

18 . The method of claim 17 , wherein the index comprises a moiety for controlled release of label.

19 . The method of claim 1 , wherein:

the step of combining the template particles with the plurality of disaggregated cells comprises combining the particles and cells in a first fluid and adding a second fluid to the first fluid, wherein the first fluid and second fluid are immiscible;

the step of generating a plurality of uniform partitions near-instantly comprises: agitating the fluids to generate the PIPs that contain a single one of the template particles and a single one of the disaggregated cells; and/or

the step of releasing in each PIP nucleic acid molecules from the cells comprises: lysing each of the single cells contained within the PIPs.

20 . The method claim 19 , wherein the step of combining cells and template particles and the step of generating a plurality of uniform partitions near-instantly are performed in a tube, wherein the tube is a centrifuge, microcentrifuge, or polymerase chain reaction (PCR) tube.

21 . The method of claim 20 , wherein the step of generating in the tube a plurality of uniform partitions near-instantly that encapsulate a single one of the template particles and a single one of the cells comprises generating a plurality of partitions that encapsulate a single one of the template particles and do not encapsulate a cell.

Assignments (2)

Continuity (2)

References Cited (211)