Single cell secretome analysis
AssigneeBecton, Dickinson and Company
InventorsHye-Won Song, Jody Martin
Life of this patent
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Abstract
Systems, methods, compositions, and kits for measuring secreted factors from cells are disclosed herein, including those capable of determining single cell secretion activity and protein expression and/or gene expression simultaneously. Disclosed herein include solid supports comprising a plurality of capture probes capable of specifically binding to at least one of the plurality of secreted factors secreted by a single cell. Also disclosed herein include secreted factor-binding reagents capable of specifically binding to a secreted factor bound by a capture probe. A secreted factor-binding reagent can comprise a secreted factor-binding reagent specific oligonucleotide comprising a unique factor identifier sequence for the secreted factor-binding reagent.
Claims24
A method for measuring the number of copies of a secreted factor secreted by a single cell, comprising:
- contacting one or more single cells with a first plurality of first solid supports, wherein the one or more single cells are capable of secreting a plurality of secreted factors, wherein each first solid support comprises a plurality of capture probes capable of specifically binding to at least one of the plurality of secreted factors secreted by a single cell;
- contacting the first solid support with a plurality of secreted factor-binding reagents each capable of specifically binding to a secreted factor bound by a capture probe, wherein each of the plurality of secreted factor-binding reagents comprises a secreted factor-binding reagent specific oligonucleotide comprising a unique factor identifier sequence for the secreted factor-binding reagent;
- contacting a plurality of oligonucleotide barcodes with the secreted factor-binding reagent specific oligonucleotides for hybridization, wherein the oligonucleotide barcodes each comprise a first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the secreted factor-binding reagent specific oligonucleotides to generate a plurality of barcoded secreted factor-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique factor identifier sequence and the first molecular label;
- and
- obtaining sequence information of the plurality of barcoded secreted factor-binding reagent specific oligonucleotides, or products thereof, to determine the number of copies of the at least one secreted factor secreted by each of the one or more single cells.
A method for measuring the number of copies of a secreted factor secreted by a single cell and the number of copies of a nucleic acid target in a single cell, comprising:
- contacting one or more single cells with a first plurality of second solid supports to form one or more single cells associated with a second solid support, wherein the one or more single cells comprise a surface cellular target and copies of a nucleic acid target, wherein the one or more single cells are capable of secreting a plurality of secreted factors, wherein each second solid support comprises a plurality of capture probes and a plurality of anchor probes, wherein each of the plurality of anchor probes is capable of specifically binding to the surface cellular target, and wherein the capture probe is capable of specifically binding to at least one of the plurality of secreted factors secreted by a single cell;
- contacting the one or more single cells associated with a second solid support with a plurality of secreted factor-binding reagents capable of specifically binding to a secreted factor bound by a capture probe, wherein each of the plurality of secreted factor-binding reagents comprises a secreted factor-binding reagent specific oligonucleotide comprising a unique factor identifier sequence for the secreted factor-binding reagent;
- contacting a plurality of oligonucleotide barcodes with the secreted factor-binding reagent specific oligonucleotides and the copies of the nucleic acid target for hybridization, wherein the oligonucleotide barcodes each comprise a first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the copies of a nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to at least a portion of the nucleic acid target and the first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the secreted factor-binding reagent specific oligonucleotides to generate a plurality of barcoded secreted factor-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique factor identifier sequence and the first molecular label;
- obtaining sequence information of the plurality of barcoded nucleic acid molecules, or products thereof, to determine the copy number of the nucleic acid target in each of the one or more single cells;
- and
- obtaining sequence information of the plurality of barcoded secreted factor-binding reagent specific oligonucleotides, or products thereof, to determine the number of copies of the at least one secreted factor secreted by each of the one or more single cells.
A composition comprising:
- a first solid support comprising a plurality of capture probes capable of specifically binding to at least one of a plurality of secreted factors secreted by a single cell;
- and
- a plurality of secreted factor-binding reagents each capable of specifically binding to a secreted factor bound by a capture probe, wherein each of the plurality of secreted factor-binding reagents comprises a secreted factor-binding reagent specific oligonucleotide comprising a unique factor identifier sequence for the secreted factor-binding reagent.
Dependencies are inferred from the claim text.