Methods and compositions for multiplex cell analysis
Provided herein in some aspects are methods, compositions, kits, and systems for performing multiplexed single-cell analysis on an in situ platform, providing alternatives which have a higher cell throughput and/or lower cost per cell compared to current single-cell analysis techniques. In some embodiments, the methods disclosed herein comprise using labeling agents that comprise sample-specific barcodes and/or cell feature specific barcodes (e.g., analyte specific barcodes) to label single-cell populations, immobilizing the labeled cells, and performing in situ detection of the labeling agents and/or other features including cellular analytes of the labeled cells.
1 . A method for sample analysis, comprising:
(a) contacting a first sample of dissociated cells with a first labeling agent and a second sample of dissociated cells with a second labeling agent,
wherein the first labeling agent and the second labeling agent each comprises (i) a binding moiety that binds to cells of the first sample and cells of the second sample, respectively, and (ii) a reporter oligonucleotide comprising a first barcode region comprising a sample-specific barcode sequence that identifies the sample origin of the cells as the first sample and the second sample, respectively
(b) combining cells from the first sample that are labeled with the first labeling agent and cells from the second sample that are labeled with the second labeling agent to provide combined labeled cells;
(c) immobilizing at least a portion of the combined labeled cells on a substrate to provide immobilized dissociated cells;
(d) contacting the immobilized dissociated cells with
a probe that directly or indirectly binds to an analyte in one or more of the immobilized dissociated cells, wherein the probe comprises a second barcode region that identifies the analyte; and
(e) analyzing (i) the sample-specific barcode sequence or a complement thereof and (ii) the second barcode region or a complement thereof in the immobilized dissociated cells on the substrate using nucleic acid sequencing or probe hybridization.
2 . The method of claim 1 , wherein the sample-specific barcode sequence and the second barcode region are analyzed by sequencing-by-synthesis.
3 . The method of claim 1 , wherein the binding moiety of the first labeling agent or the binding moiety of the second labeling agent covalently binds to one or more molecules in or on a cell of the first sample or the second sample, respectively.
4 . The method of claim 1 , wherein the binding moiety of the first labeling agent or the binding moiety of the second labeling agent noncovalently binds to one or more molecules in or on a cell of the first sample or the second sample, respectively.
5 . The method of claim 1 , wherein the binding moiety of the first labeling agent or the binding moiety of the second labeling agent binds to a surface of a cell of the first sample or the second sample, respectively.
6 . The method of claim 1 , wherein the binding moiety of the first labeling agent or the binding moiety of the second labeling agent is selected from the group consisting of: an antibody or an epitope binding fragment thereof; a lipophilic moiety; a receptor; a receptor ligand; a small molecule; an aptamer; a monobody; an affimer; a darpin; and a protein scaffold.
7 . The method of claim 1 , wherein the analyte is a nucleic acid analyte.
8 . The method of claim 1 , wherein the analyte is a polypeptide.
9 . The method of claim 1 , wherein the immobilized dissociated cells comprise at least 50,000 dissociated cells.
10 . The method of claim 1 , wherein the first labeling agent or the second labeling agent further comprises a target-specific barcode sequence that identifies the binding moiety in the first labeling agent or the second labeling agent, respectively.
11 . The method of claim 1 , wherein the binding moiety is an antibody.
12 . The method of claim 1 , wherein the analyte is a cellular RNA.
13 . The method of claim 12 , wherein the probe is a first circularizable probe that hybridizes to the cellular RNA.
14 . The method of claim 13 , further comprising generating, on the substrate, a first rolling circle amplification (RCA) product of a first circularized probe, wherein the first circularized probe is generated from the first circularizable probe hybridized to the cellular RNA.
15 . The method of claim 14 , further comprising analyzing the first RCA product on the substrate using (i) sequencing-by-synthesis or (ii) a plurality of detectable probes that hybridize to the first RCA product.
16 . The method of claim 14 , further comprising contacting the immobilized dissociated cells on the substrate with a second circularizable probe that hybridizes to the sample-specific barcode sequence.
17 . The method of claim 16 , further comprising generating, on the substrate, a second RCA product of a second circularized probe, wherein the second circularized probe is generated from the second circularizable probe hybridized to the sample-specific barcode sequence.
18 . The method of claim 17 , further comprising analyzing the second RCA product on the substrate using (i) sequencing-by-synthesis or (ii) a plurality of detectable probes that hybridize to the second RCA product.
19 . The method of claim 1 , wherein in (c), at least 100 cells are immobilized within an area of 1 cm 2 on the substrate.
20 . The method of claim 1 , wherein in (c), at least 100,000 cells are immobilized within an area of 1 cm 2 on the substrate.