METHODS FOR IN SITU TRANSCRIPTOMICS AND PROTEOMICS
Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a cell.
1 .- 30 . (canceled)
31 . A method of detecting a plurality of targets comprising different proteins within an optically resolved volume of a cell in situ; said method comprising:
i) contacting each of the targets with a specific binding reagent, wherein the specific binding reagent comprises an oligonucleotide barcode;
ii) hybridizing a padlock probe to two nucleic acid sequences of the barcode, wherein the padlock probe is a single-stranded polynucleotide having a 5′ and a 3′ end, wherein the padlock probe comprises a primer binding sequence from a known set of primer binding sequences;
iii) sequencing each barcode to obtain a multiplexed signal in the cell in situ;
iv) demultiplexing the multiplexed signal by comparison with the known set of barcodes; and
v) detecting the plurality of targets by identifying the associated barcodes detected in the cell.
32 . The method of claim 31 , wherein the specific binding reagent comprises an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), or an aptamer.
33 . The method of claim 31 , wherein hybridizing the padlock probe comprises hybridizing the 3′ end of the padlock probe to a first complementary region of the barcode, and hybridizing the 5′ end of the padlock probe to a second complementary region of the barcode, wherein the second complementary region is adjacent to the first complementary region.
34 . The method of claim 31 , wherein hybridizing the padlock probe comprises hybridizing the 3′ end of the padlock probe to a first complementary region of the barcode, and hybridizing the 5′ end of the padlock probe to a second complementary region of the barcode, wherein the second complementary region is about 5 or more nucleotides in the 5′ direction with respect to the first complementary region.
35 . The method of claim 34 , wherein the first complementary region and the second complementary region of the barcode are separated by about 5 to about 75 nucleotides.
36 . The method of claim 31 , further comprising ligating the 5′ and 3′ ends of the padlock probe to form a circular polynucleotide.
37 . The method of claim 36 , wherein the circular polynucleotide comprises a sequence of the barcode, or a complement thereof.
38 . The method of claim 36 , further comprising amplifying the circular polynucleotide by extending an amplification primer with a strand-displacing polymerase, wherein the primer extension generates an extension product comprising multiple complements of the circular polynucleotide.
39 . The method of claim 31 , wherein the known set of primer binding sequences comprises at least 2 different primer binding sequences.
40 . The method of claim 31 , wherein the barcode is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.
41 . The method of claim 31 , wherein the number of unique targets detected within an optically resolved volume of the cell is about 3, 10, 30, 50, or 100.
42 . The method of claim 31 , wherein sequencing comprises (a) extending a sequencing primer by incorporating a labeled nucleotide, or labeled nucleotide analogue and (b) detecting the label to generate a signal for each incorporated nucleotide or nucleotide analogue.
43 . The method of claim 42 , wherein the labeled nucleotide or labeled nucleotide analogue further comprises a reversible terminator moiety.
44 . The method of claim 31 , wherein the padlock probe further comprises a second oligonucleotide barcode, wherein each oligonucleotide barcode is from a known set of barcodes associated with each of the plurality of targets.
45 . The method of claim 31 , wherein the cell is permeabilized and immobilized to a solid support surface.
46 . The method of claim 45 , wherein the surface comprises a patterned surface suitable for immobilization of a plurality of cells in an ordered pattern.
47 . The method of claim 31 , wherein the method does not comprise lysing the cell.
48 . The method of claim 31 , wherein barcodes in the known set of barcodes have a specified Hamming distance, wherein the Hamming distance is 1 to 15.
49 . The method of claim 31 , wherein the cell forms part of a tissue in situ.
50 . The method of claim 31 , wherein the method is performed simultaneously in at least 1000 cells.