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 RNA molecules within cells, the method comprising:
 (a) fixing and permeabilizing a plurality of cells;   (b) generating complementary DNA (cDNA) molecules within the plurality of cells by reverse transcribing RNA molecules within the cells, wherein the RNA molecules are reverse transcribed using reverse transcription (RT) primers each comprising:
 a poly(T) sequence or a random nucleotide sequence, and 
 a 5′ overhang comprising a 5′ overhang sequence; and 
   (c) tagging the cDNA molecules with one or more barcode sequences by performing steps (i) through (iii) one or more times:
 (i) dividing the plurality of cells comprising the cDNA molecules into a plurality of aliquots, wherein each aliquot comprises more than one cell; 
 (ii) coupling nucleic acid tags to cDNA molecules within cells of the aliquots, wherein each nucleic acid tag comprises a barcode sequence, and wherein the barcode sequences present within the nucleic acid tags are specific to each aliquot; and 
 (iii) combining the cells from the plurality of aliquots. 
   
     
     
         2 . The method of  claim 1 , further comprising:
 (d) dividing the combined cells from the plurality of aliquots into a plurality of samples;   (e) lysing the cells in the plurality of samples to release the cDNA molecules; and   (f) amplifying the released cDNA molecules in the plurality of samples using one or more amplification primers, wherein at least one of the amplification primers used in each sample comprises an index sequence, and wherein the index sequences present within the amplification primers are specific to each sample.   
     
     
         3 . The method of  claim 1 , wherein steps (i) through (iii) are repeated 1, 2, 3, 4, or 5 times. 
     
     
         4 . The method of  claim 1 , wherein steps (i) through (iii) are repeated a number of times sufficient to generate at least as many unique barcode sequence combinations as the total number of cells in the plurality of cells. 
     
     
         5 . The method of  claim 1 , wherein steps (i) through (iii) are repeated a number of times sufficient to provide a greater than 50%, 90%, 95%, or 99% probability that the cDNA molecules in each cell are bound to a unique barcode sequence combination. 
     
     
         6 . The method of  claim 1 , wherein the nucleic acid tags each comprise a 3′ region and/or a 5′ region flanking the barcode sequence. 
     
     
         7 . The method of  claim 1 , wherein the coupling comprises ligating 3′ ends of the nucleic acid tags to 5′ ends of the cDNA molecules. 
     
     
         8 . The method of  claim 7 , wherein the 3′ ends of the nucleic acid tags are brought into proximity of the 5′ ends of the cDNA molecules by prehybridizing the nucleic acid tags with linker nucleic acid strands that are each complementary to a 3′ terminal sequence of a nucleic acid tag and a 5′ terminal sequence of a cDNA molecule. 
     
     
         9 . The method of  claim 8 , wherein the 5′ terminal sequence of the cDNA molecule comprises a 5′ overhang sequence from an RT primer or a 5′ region from a previously coupled nucleic acid tag. 
     
     
         10 . The method of  claim 8 , wherein the coupling is stopped by introducing a plurality of ligation stop oligos that are each complementary to all or part of a linker nucleic acid strand. 
     
     
         11 . The method of  claim 1 , wherein the nucleic acid tags are DNA tags. 
     
     
         12 . The method of  claim 1 , wherein the nucleic acid tags that are coupled to the cDNA molecules during the last of the one or more times comprise a unique molecular identifier (UMI), a capture agent, a flow-cell binding site, and/or a primer-binding site. 
     
     
         13 . The method of  claim 13 , wherein the capture agent comprises biotin. 
     
     
         14 . The method of  claim 1 , wherein the barcode sequences each comprise at least 8 nucleotides. 
     
     
         15 . 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. 
     
     
         16 . The method of  claim 1 , wherein all of the RT primers comprise a poly(T) sequence. 
     
     
         17 . The method of  claim 1 , wherein all of the RT primers comprise a random nucleotide sequence. 
     
     
         18 . The method of  claim 2 , further comprising:
 (g) sequencing the cDNA molecules amplified in step (f).   
     
     
         19 . The method of  claim 18 , further comprising:
 (h) grouping the sequencing reads obtained in step (g) by barcode sequence and/or index sequence.   
     
     
         20 . The method of  claim 19 , wherein the sequencing reads are grouped by a combination of barcode sequence and index sequence. 
     
     
         21 . The method of  claim 20 , wherein grouping by barcode sequence comprises grouping by barcode sequence combination. 
     
     
         22 . The method of  claim 1 , wherein the plurality of aliquots comprises 96 aliquots distributed in a 96-well plate.