	US 12,071,656 B2
	Methods and compositions for identifying or quantifying targets in a biological sample
Marlon Stoeckius, New York, NY (US); Peter Smibert, New York, NY (US); and Brian Houck-Loomis, New York, NY (US)
Assigned to New York Genome Center, Inc., New York, NY (US)
Filed by New York Genome Center, Inc., New York, NY (US)
Filed on Apr. 30, 2021, as Appl. No. 17/245,479.
Application 17/245,479 is a continuation of application No. 15/887,144, filed on Feb. 2, 2018, abandoned.
Claims priority of provisional application 62/609,332, filed on Dec. 21, 2017.
Claims priority of provisional application 62/599,450, filed on Dec. 15, 2017.
Claims priority of provisional application 62/559,228, filed on Sep. 15, 2017.
Claims priority of provisional application 62/549,189, filed on Aug. 23, 2017.
Claims priority of provisional application 62/515,180, filed on Jun. 5, 2017.
Claims priority of provisional application 62/453,726, filed on Feb. 2, 2017.
Prior Publication US 2021/0371914 A1, Dec. 2, 2021
Int. Cl. C12Q 1/68 (2018.01); C12N 15/10 (2006.01); C12Q 1/6804 (2018.01); C12Q 1/6844 (2018.01); C40B 50/06 (2006.01)

CPC C12Q 1/6844 (2013.01) [C12N 15/1093 (2013.01); C12Q 1/6804 (2013.01); C40B 50/06 (2013.01)]

8 Claims

1. A method of simultaneously identifying from a single sequencing run a transcriptome and one or more proteins on one or more cells which comprise cellular mRNA, the method comprising:

a) providing the one or more cells originating from a biological sample, wherein each of the one or more cells comprises one or more epitopes, and each of the one or more epitopes corresponds to one of the one or more proteins on the one or more cells;

b) generating one or more complexes, wherein each of the one or more generated complexes comprises:

(i) one of the one or more cells;

(ii) a first construct comprising:

(a) a first antibody or fragment thereof that specifically binds to a first of the one or more epitopes; and,

(b) a first construct oligonucleotide, the first construct oligonucleotide conjugated to the first antibody or fragment thereof by a linker,

wherein the first construct oligonucleotide comprises:

(i) an amplification handle;

(ii) a first antibody barcode sequence that specifically identifies said first antibody or fragment thereof from any other antibody or fragment thereof that recognizes a different epitope of the one or more epitopes; and,

(iii) an anchor sequence;

and,

(iii) one or more distinct additional constructs, each distinct additional construct comprising:

(a) an additional antibody or fragment thereof that specifically binds to an additional epitope of the one or more epitopes; and,

(b) an additional construct oligonucleotide, wherein each additional construct oligonucleotide is conjugated to the additional antibody or fragment thereof by a linker,

wherein each of the additional construct oligonucleotides comprises:

(i) an amplification handle;

(ii) an additional antibody barcode sequence that specifically identifies said additional antibody or fragment thereof from any other antibody or fragment that recognizes a different epitope of the one or more epitopes; and,

(iii) an anchor sequence,

wherein each of the additional one or more epitopes is different from the first epitope and the additional epitope of the one or more epitopes of the other distinct additional constructs, and each of the distinct additional constructs differs from the corresponding components of the first construct and the other distinct additional constructs by the additional antibody or fragment thereof and the additional antibody barcode sequence;

c) individually partitioning each of the generated one or more complexes with a bead, each bead conjugated to capture oligonucleotides that comprise:

(i) sequences that hybridize to the anchor sequences; and,

(ii) a bead-specific barcode sequence unique to each bead;

d) lysing the one or more cells to release:

(i) the first construct;

(ii) the one or more distinct additional constructs; and,

(iii) the cellular mRNA present in the one or more cells;

e) annealing:

(i) the cellular mRNA to the capture oligonucleotides; and,

(ii) the first construct oligonucleotide and the additional construct oligonucleotides to the capture oligonucleotides;

f) generating:

(i) cDNA from the annealed cellular mRNA and the capture oligonucleotides; and

(ii) DNA from the annealed first construct oligonucleotide, the additional construct oligonucleotides, and the capture oligonucleotides;

g) substantially separating by size:

(i) the cDNA generated from the annealed cellular mRNA and the capture oligonucleotides; and,

(ii) the DNA generated from the annealed first construct oligonucleotide, the additional construct oligonucleotides, and the capture oligonucleotides;

h) generating, independently from each other:

(i) a transcriptome amplification library comprising cDNAs amplified from the cDNAs generated from the annealed cellular mRNA and the capture oligonucleotides; and,

(ii) a protein amplification library comprising DNAs amplified from the DNAs generated from the annealed first construct oligonucleotide, the additional construct oligonucleotides, and the capture oligonucleotides;

i) pooling the transcriptome amplification library and the protein amplification library after they are generated;

j) sequencing, in parallel, the pooled transcriptome amplification library and the protein amplification library, in a single sequencing run; and,

k) from the parallel sequencing, using the antibody barcode sequences and the bead-specific barcode sequences to simultaneously identify the transcriptome and the one or more proteins on the one or more cells.