Single-cell rna sequencing using click-chemistry

Abstract

The present disclosure relates to a method of sequencing nascent RNA in a cell. In some embodiments, the nascent RNA is conjugated to DNA using copper-catalyzed azide-alkyne cycloaddition (CuAAC). Methods of the present disclosure can be used to generate genomic libraries of a cell and measure gene expression and enhancer and/or super-enhancer activity.

1 .- 47 . (canceled) 
     
     
         48 . A method for locating enhancers and/or super-enhancers in a permeabilized cell or nucleus comprising
 (i) oxidizing 3′-terminal cis-diol groups of RNA in the permeabilized cell or nucleus to dialdehyde and then conjugating an alkyne group to the dialdehyde by reductive amination by contacting the dialdehyde with a primary amine to form an imine intermediate and reducing the imine to amine using a reducing reagent; or   (ii) transferring an azide or an alkyne group to the gamma (γ) phosphate on the 5′ end of RNAs in the permeabilized cell or nucleus;   isolating the RNAs of (i) or (ii);   sequencing the isolated RNAs; and   (a) aligning the sequenced isolated RNAs to a reference genome, wherein enhancers and/or super-enhancers are located upstream or downstream of transcribed genes; or   (b) measuring the level of RNAs by quantitative PCR (qPCR).   
     
     
         49 . The method of  claim 48 , wherein the reducing reagent in (i) is cyanoborohydride, borohydride, formic acid, or palladium on carbon (PD/c). 
     
     
         50 . (canceled) 
     
     
         51 . The method of  claim 48 , wherein the transferring in (ii) is catalyzed by polynucleotide kinase or by ligation or hybridization of a small oligonucleotide containing azide or alkyne. 
     
     
         52 .- 57 . (canceled) 
     
     
         58 . The method of  claim 48 , wherein a higher level of the enhancer and/or super-enhancer activity results in increased RNAs from the genes regulated by the enhancer and/or super-enhancer relative to a control. 
     
     
         59 . The method of  claim 48 , wherein a lower level of the enhancer and/or super-enhancer activity results in decreased RNAs from the genes regulated by the enhancer and/or super-enhancer relative to a control. 
     
     
         60 . A method of generating a library of nascent RNA sequences in a permeabilized cell or nucleus or a plurality of permeabilized cells or nuclei comprising:
 (i) oxidizing a 3′-terminal cis-diol group of nascent RNAs in the permeabilized cell or nucleus or the plurality of permeabilized cells or nuclei to dialdehyde and then conjugating an alkyne group to the dialdehyde by reductive amination by contacting the dialdehyde with a primary amine to form an imine intermediate and reducing the imine to amine using a reducing reagent to form labeled RNAs; or   (ii) transferring an azide or an alkyne group to the gamma (γ) phosphate on the 5′ end of RNAs in the permeabilized cell or nucleus or the plurality of permeabilized cells or nuclei;   isolating the labeled RNAs of (i) or (ii); and   conjugating the isolated RNAs to a bead.   
     
     
         61 .- 71 . (canceled) 
     
     
         72 . A method for sequencing of nascent RNA in a single permeabilized cell or nucleus comprising
 isolating a single permeabilized cell or nucleus;   labeling nascent RNA in the single permeabilized cell or nucleus by incubating the permeabilized cell or nucleus with either alkyne-labeled NTPs or azide-NTPs, which are incorporated into nascent RNA transcripts by RNA polymerase in the single permeabilized cell or nucleus;   contacting a lysate of the single permeabilized cell or nucleus with azide-labeled single-cell barcode-adaptors or alkyne-labeled single-cell barcode-adaptors in the presence of a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction reagent, forming RNA-single-cell-barcoded-adaptor conjugates;   fragmenting the nascent RNA by incubating with zinc chloride at 65° C.;   reverse transcribing the nascent RNA into complementary DNA (cDNA); and   sequencing the cDNA.   
     
     
         73 . The method of  claim 72 , further comprising amplifying the cDNA by PCR to produce a PCR product. 
     
     
         74 . The method of  claim 72  or  73 , further comprising size-selecting a PCR product by polyacrylamide gel electrophoresis. 
     
     
         75 . The method of  claim 72 , wherein the single-cell barcode-adaptors are immobilized on a bead or wherein the single-cell barcode-adaptors are free. 
     
     
         76 . (canceled) 
     
     
         77 . The method of  claim 72 , wherein the nascent RNA is sequenced from a plurality of permeabilized cells or nuclei. 
     
     
         78 . The method of  claim 72 , wherein the CuAAC reaction reagent is copper sulfate (CuSO4), tetrakis(acetonitrile)copper(I)hexafluorophosphate ((Cu(CH3CN)4]PF6), tetrakis(acetonitrile)copper(I) triflate (Cu(CH3CN)4]OTf, copper acetate (C 4 H 6 CuO 4 ), copper bromide (BrCu), or copper iodide (CuI). 
     
     
         79 . The method of  claim 72 , wherein a reducing reagent reduces Cu(II) to Cu(I) in the CuAAC reaction. 
     
     
         80 . The method of  claim 79 , wherein the reducing agent is sodium ascorbate, hydrazine, tris(2-carboxyethyl)phosphine (TCEP), dithiotreitol (DTT), or beta-mercaptoethanol. 
     
     
         81 . The method of  claim 72 , wherein the CuAAC reaction is in the presence of an accelerating ligand, optionally wherein the accelerating ligand is 2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetic acid (BTTAA), (1-(4-methoxybenzyl)-1-H-1,2,3-triazol-4-yl)methanol (MBHTM), or tris-hydroxypropyltriazolylmethylamine (THPTA). 
     
     
         82 .- 85 . (canceled) 
     
     
         86 . A method of identifying a cell type or types, the differentiation state of a cell, or the activation state of a cell comprising:
 analyzing the nascent RNAs from a single permeabilized cell or nucleus according to the method of  claim 72 ; and
 a) sequencing the isolated nascent RNAs; or 
 b) measuring the level of isolated nascent RNAs by quantitative PCR (qPCR); and 
   comparing the sequence or level of isolated nascent RNAs to control RNA samples to identify the cell type or types, the differentiation state of the cell, or the activation state of the cell.   
     
     
         87 . The method of  claim 86 , wherein the nascent RNA is sequenced from a population of permeabilized cells or nuclei. 
     
     
         88 . The method of  claim 86 , wherein the control RNA samples are from a subject having known specific cell types, from a subject known not to have specific cell types, or from a reference sequence, wherein the reference sequence is known to be associated with a particular cell type. 
     
     
         89 . (canceled) 
     
     
         90 . (canceled) 
     
     
         91 . The method of  claim 86 , wherein the control RNA samples are from a subject known to have a specific differentiation state, from a subject known to not have a specific differentiation state, or from a reference sequence, wherein the reference sequence is known to be associated with a specific differentiation state. 
     
     
         92 . (canceled) 
     
     
         93 . (canceled) 
     
     
         94 . The method of  claim 86 , wherein the control RNA samples are from a subject known to have a specific activation state, from a subject known to not have a specific activation state, or from a reference sequence, wherein the reference sequence is known to be associated with a specific activation state.