Methods and kits for labeling cellular molecules

Abstract

Methods of uniquely labeling or barcoding molecules within a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs, cDNAs, DNAs, proteins, peptides, and/or antigens.

1 . A method of cell-specifically labeling deoxyribonucleic acid (DNA) molecules within a plurality of cells, the method comprising:
 (a) providing a plurality of fixed, permeabilized cells, wherein each of the plurality of cells comprises genomic DNA;   (b) fragmenting the genomic DNA within each of the plurality of cells using one or more restriction enzymes or one or more transposases, thereby producing a plurality of DNA fragments;   (c) dividing the plurality of cells comprising the plurality of DNA fragments into a plurality of primary aliquots, wherein the plurality of primary aliquots comprises a first primary aliquot and a second primary aliquot;   (d) coupling primary nucleic acid tags to DNA fragments in cells of the primary aliquots, thereby producing primary nucleic acid-tagged DNA fragments, wherein the primary nucleic acid-tagged DNA fragments in cells of the first primary aliquot are tagged with different primary nucleic acid tags than the primary nucleic acid-tagged DNA fragments in cells of the second primary aliquot;   (e) combining the plurality of primary aliquots;   (f) dividing the combined primary aliquots of (e) into a plurality of secondary aliquots, wherein the plurality of secondary aliquots comprises a first secondary aliquot and a second secondary aliquot; and   (g) coupling secondary nucleic acid tags to primary nucleic acid-tagged DNA fragments in the secondary aliquots, thereby producing secondary nucleic acid-tagged DNA fragments, wherein the secondary nucleic acid-tagged DNA fragments of the first secondary aliquot are tagged with different secondary nucleic acid tags than the secondary nucleic acid-tagged DNA molecules of the second secondary aliquot.   
     
     
         2 . The method of  claim 1 , further comprising repeating (e), (f), and (g) one or more times. 
     
     
         3 . The method of  claim 2 , wherein (e), (f), and (g) are repeated a number of times sufficient to generate a unique series of nucleic acid tags for each of the DNA fragments in a single cell of the plurality of cells. 
     
     
         4 . The method of  claim 3 , wherein the number of times is 1, 2, 3, 4, or 5. 
     
     
         5 . The method of  claim 1 , wherein each of the primary nucleic acid tags comprises a first strand comprising a barcode sequence. 
     
     
         6 . The method of  claim 5 , wherein the first strand of the primary nucleic acid tags comprises a 3′ hybridization sequence flanking a 3′ end of the barcode sequence, or a 5′ hybridization sequence flanking a 5′ end of the barcode sequence, or both. 
     
     
         7 . The method of  claim 1 , wherein each of the secondary nucleic acid tags comprises a first strand comprising a barcode sequence. 
     
     
         8 . The method of  claim 7 , wherein the first strand of the secondary nucleic acid tags comprises a 3′ hybridization sequence flanking a 3′ end of the barcode sequence, or a 5′ hybridization sequence flanking a 5′ end of the barcode sequence, or both. 
     
     
         9 . The method of  claim 1 , wherein, prior to (c) a single-stranded adapter is coupled to each of the DNA fragments. 
     
     
         10 . The method of  claim 9 , wherein the fragmenting in (a) is performed by a transposase, and wherein the single-stranded adapters are coupled to the DNA fragments by the transposase. 
     
     
         11 . The method of  claim 9  or  claim 10 , wherein coupling the primary nucleic acid tags to the DNA fragments in (d) comprises coupling the primary nucleic acid tags to single stranded adapters coupled to the DNA fragments. 
     
     
         12 . The method of  claim 1 , wherein coupling the secondary nucleic acid tags to the primary nucleic acid-tagged DNA fragments in (g) comprises coupling the secondary nucleic acid tags to primary nucleic acid tags coupled to the DNA fragments. 
     
     
         13 . The method of  claim 1 , wherein coupling the primary nucleic acid tags to DNA fragments in (d) and/or coupling the secondary nucleic acid tags to primary nucleic acid tagged DNA fragments in (g) comprises ligation that takes place within the plurality of cells. 
     
     
         14 . The method of  claim 1 , further comprising removing primary nucleic acid tags that are unbound to DNA fragments from cells prior to (e) or (f) and/or removing secondary nucleic acid tags that are unbound to DNA fragments from cells subsequent to (g). 
     
     
         15 . The method of  claim 2 , wherein the nucleic acid tags that are coupled to the DNA fragments during the last of the one or more times that steps (e) to (g) are repeated comprise a capture agent. 
     
     
         16 . The method of  claim 1 , further comprising: (h) lysing the plurality of cells to release the DNA fragments from within the plurality of cells. 
     
     
         17 . The method of  claim 16 , wherein coupling the primary nucleic acid tags to DNA fragments in (e) and/or coupling the secondary nucleic acid tags to the primary nucleic acid tagged DNA fragments in (g) comprise hybridizing the nucleic acid tags to the DNA fragments, and further comprising: (i) ligating the hybridized nucleic acid tags to the DNA fragments. 
     
     
         18 . The method of  claim 17 , wherein a majority of the nucleic acid-tagged DNA fragments from a single cell comprise the same combination of primary and secondary nucleic acid tags coupled thereto. 
     
     
         19 . The method of  claim 1 , wherein the plurality of cells is selected from the group consisting of mammalian cells, yeast cells, bacterial cells, and combinations thereof. 
     
     
         20 . The method of  claim 6 , wherein prior to (c) a single-stranded adapter is coupled to a 5′ end of each of the DNA fragments, and wherein the primary nucleic acid tags each further comprise a second linker nucleic acid strand comprising: a first portion complementary to the 5′ end of the single-stranded adapter, and a second portion complementary to the 3′ hybridization sequence of the first strand of the primary nucleic tag. 
     
     
         21 . The method of  claim 8 , wherein the first strand of the primary nucleic acid tags each comprises a 5′ hybridization sequence flanking a 5′ end of the barcode sequence, and wherein the secondary nucleic acid tags each further comprise a second linker nucleic acid strand comprising: a first portion complementary to a 5′ hybridization sequence of a previously coupled primary nucleic acid tag, and a second portion complementary to the 3′ hybridization sequence of the first strand of secondary nucleic acid tag. 
     
     
         22 . The method of  claim 20  or  21 , wherein coupling the primary nucleic acid tags to DNA fragments in step (e) and/or coupling the secondary nucleic acid tags to primary nucleic acid tagged DNA fragments in (g) comprise ligation that is performed within the plurality of cells, and wherein the method further comprises stopping the coupling in (e) by introducing a plurality of ligation stop oligos complementary to all or part of the second linker nucleic acid strand of the primary nucleic acid tag, and/or stopping the coupling in (g) by introducing a plurality of ligation stop oligos complementary to all or part of the second linker nucleic acid strand of the secondary nucleic acid tag. 
     
     
         23 . The method of  claim 1 , wherein the plurality of cells is fixed using formaldehyde. 
     
     
         24 . A method of cell-specifically labeling DNA molecules within a plurality of cells, the method comprising:
 (a) providing a plurality of fixed, permeabilized cells, wherein each of the plurality of cells comprises genomic DNA;   (b) fragmenting the genomic DNA with one or more restriction enzymes or one or more transposases and tagging the DNA molecules, thereby producing a plurality of tagged DNA fragments, wherein the tagged DNA fragments comprise an adaptor;   (c) dividing the plurality of cells comprising the tagged DNA fragments into a plurality of primary aliquots, wherein the plurality of primary aliquots comprises a first primary aliquot and a second primary aliquot;   (d) coupling primary nucleic acid tags to the tagged DNA fragments in each of the plurality of primary aliquots, thereby producing primary nucleic acid-tagged DNA molecules, wherein the primary nucleic acid-tagged DNA molecules of the first primary aliquot are tagged with different primary nucleic acid tags than the primary nucleic acid-tagged DNA molecules of the second primary aliquot;   (e) combining the plurality of primary aliquots;   (f) dividing the combined primary aliquots of (e) into a plurality of secondary aliquots, wherein the plurality of secondary aliquots comprises a first secondary aliquot and a second secondary aliquot; and   (g) coupling secondary nucleic acid tags to the primary nucleic acid-tagged DNA molecules, thereby producing secondary nucleic acid-tagged DNA molecules, wherein the secondary nucleic acid-tagged DNA molecules of the first secondary aliquot are tagged with different secondary nucleic acid tags than the secondary nucleic acid-tagged DNA molecules of the second secondary aliquot.