Methods and systems for performing single cell analysis of molecules and molecular complexes

Abstract

Methods, systems and related compositions are provided to perform single-cell marking of a nucleic acid and/or protein in a sample based on in-cell or in-organelle barcoding of nucleic acid and/or protein complexes of the cell or organelle; the methods and systems herein described are configured to provide in-cell or in-organelle single-cell marked nucleic acid and/or protein complexes comprising a single-cell, cell-specific, or a single-cell organelle-specific marker.

1 . A method to perform single-cell marking of a nucleic acid and/or protein in a sample comprising a plurality of cells, the method comprising:
 crosslinking a cell from the plurality of cells to provide a crosslinked cell comprising a crosslinked nucleic acid and/or protein material,   permeabilizing the crosslinked cell from the plurality of cells or a nucleus thereof, to provide a permeabilized cell or nucleus thereof;   in-cell or in-nucleus fragmenting the crosslinked nucleic acid and/or protein material to provide molecular complexes each comprising fragmented crosslinked nucleic acid and/or protein material   in-cell or in-nucleus barcoding nucleic acid and/or protein in the molecular complexes of the permeabilized cell or nucleus thereof, following the in-cell or in-nucleus fragmenting,   to provide an in-cell or in-nucleus single-cell or single-nucleus marked-nucleic acid and/or protein in the molecular complexes of the cell, the nucleic acids and/or proteins barcoded with a single-cell or single-nucleus specific marker.   
     
     
         2 . The method of  claim 1 , wherein the in-cell or in-nucleus barcoding is performed by sequentially attaching a series of tags to provide a barcode formed by a series of two or more tags directly attached one to another through covalent linkage. 
     
     
         3 . The method of  claim 1 , wherein the in-cell or in-nucleus barcoding is performed without performing an enzymatic intracomplex ligation step directed to include a covalent linkage between two nucleic acids and/or proteins which are attached to one another within a complex. 
     
     
         4 . The method of  claim 1 , wherein
 the permeabilizing is performed by permeabilizing the nucleus thereof; and   the in-cell or in-nucleus barcoding is performed by in-nucleus attaching a series of tags to the nucleic acid and/or protein within the molecular complexes of the permeabilized nucleus thereof, to provide a single-cell marked nucleic acid and/or protein within the molecular complexes comprising a single-cell nucleus specific marker.   
     
     
         5 . The method of  claim 4 , wherein the method further comprises, before the permeabilizing
 isolating the nucleus thereof from the cell from the plurality of cells.   
     
     
         6 .- 7 . (canceled) 
     
     
         8 . The method of  claim 4 , wherein the method further comprises, before the crosslinking
 isolating the nucleus thereof from the cell.   
     
     
         9 . The method of  claim 1 , wherein the method further comprises, before the crosslinking
 isolating the cell from the plurality of cells.   
     
     
         10 . The method of  claim 9 , wherein the method further comprises, after the isolating the cell and before the crosslinking:
 isolating the nucleus thereof from the cell.   
     
     
         11 . The method of  claim 4 , wherein the nucleus is a single nucleus from a single cell of the plurality of cells and the in-nucleus barcoding is performed by ligating a barcode comprising a single tag to the nucleic acid and/or protein complexes. 
     
     
         12 . The method of  claim 4 , wherein the nucleus is a plurality of nuclei each from a single cell of the plurality of cells and the in-nucleus attaching a series of tags is performed in-nucleus split and pool barcoding the nucleic acid and/or protein complexes of each organelle of the plurality of organelles. 
     
     
         13 . The method of  claim 4 , further comprising, after the in-nucleus barcoding,
 lysing the organelle to isolate single-cell marked nucleic acid and/or protein complexes comprising a single-cell nucleus-specific marker from the nucleus.   
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein
 the permeabilizing is performed by permeabilizing the cell; and   the in-cell or in-nucleus barcoding is performed by in-cell barcoding the nucleic acid and/or protein within the molecular complexes of the permeabilized cell, to provide single-cell marked nucleic acid and/or protein complexes comprising a single-cell-specific marker.   
     
     
         16 . The method of  claim 15 , wherein the method further comprises, before the permeabilizing
 isolating the cell from the plurality of cell.   
     
     
         17 .- 18 . (canceled) 
     
     
         19 . The method of  claim 15 , wherein the method further comprises, before the crosslinking:
 isolating the cell from the plurality of cells.   
     
     
         20 . The method of  claim 15 , wherein the cell is a single cell of the plurality of cells and the in-cell barcoding is performed by ligating a barcode comprising a single tag to the nuclei acid and/or protein within the molecular complexes of the permeabilized cell. 
     
     
         21 . The method of  claim 15 , wherein the cell is a plurality of cells and the in-cell barcoding is performed by in-cell split and pool barcoding the nucleic acid and/or protein complexes of each cell. 
     
     
         22 . The method of  claim 15 , further comprising after the in-cell barcoding:
 lysing the cell to isolate single-cell marked nucleic acid and/or protein within the molecular complexes comprising a single-cell cell specific tag from the cell.   
     
     
         23 .- 27 . (canceled) 
     
     
         28 . The method of  claim 1  wherein the in-cell or in-nucleus attaching a series of tags is performed by in-cell or in-nucleus attaching a series of tags to a nucleic acid of nucleic acid complexes and/or nucleic acid protein complexes. 
     
     
         29 . The method of  claim 1  wherein the in-cell or in-nucleus attaching a series of tags is performed by in-cell or in-nucleus attaching a series of tags to a protein of protein complexes and/or nucleic acid protein complexes. 
     
     
         30 . The method of  claim 1 , wherein the in-cell or in-nucleus attaching a series of tags is performed by attaching a series of tags to a ribonucleic acid of the molecular complexes. 
     
     
         31 . The method of  claim 30 , wherein the in-cell or in-nucleus barcoding is performed by sequentially attaching a series of tags to provide a barcode formed by a series of two or more tags directly attached one to another through covalent linkage. 
     
     
         32 . The method of  claim 30 , wherein the cell or nucleus thereof is a plurality of cells or nuclei thereof and the in-cell or in-nucleus barcoding is performed by in-cell or in-nucleus split and pool barcoding of the ribonucleic acid of the molecular complexes of each cells of the plurality of cells or of each nucleus thereof. 
     
     
         33 . The method of  claim 30 , further comprising,
 lysing the nucleus to isolate single-cell marked molecular complexes comprising a nucleic acid and/or protein barcoded with a single-cell nucleus specific marker from the nucleus, the lysing performed after the in-nucleus barcoding.   
     
     
         34 . The method of  claim 30 , wherein
 the permeabilizing is performed by permeabilizing the nucleus thereof; and   the in-cell or in-nucleus barcoding is performed by in-nucleus barcoding the nucleic acid and/or protein within the molecular complexes of the permeabilized organelle thereof,
 to provide single-nucleus marked molecular complexes comprising ribonucleic acid barcoded with a single-nucleus specific marker. 
   
     
     
         35 . The method of  claim 34 , wherein the method further comprises,
 isolating the nucleus thereof from the cell the isolating performed before the crosslinking.   
     
     
         36 . The method of  claim 34 , wherein the nucleus thereof is a single nucleus from a single cell of the plurality of cells. 
     
     
         37 . The method of  claim 36 , wherein the method further comprises,
 isolating the nucleus thereof from the cell, the isolating performed after isolating the cell and before the crosslinking.