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EP-4741512-A2 - IN SITU COMBINATORIAL LABELING OF CELLULAR MOLECULES

EP4741512A2EP 4741512 A2EP4741512 A2EP 4741512A2EP-4741512-A2

Abstract

Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, 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 and/or cDNAs.

Inventors

  • SEELIG, GEORG
  • ROSENBERG, ALEXANDER B.
  • ROCO, Charles

Assignees

  • University of Washington

Dates

Publication Date
20260513
Application Date
20180921

Claims (13)

  1. A method of uniquely labeling RNA molecules within a plurality of nuclei, the method comprising: (a) fixing and permeabilizing a first plurality of nuclei prior to step (b), wherein the first plurality of nuclei are fixed and permeabilized at 4 °C or below 4 °C; (b) reverse transcribing the RNA molecules within the first plurality of nuclei to form complementary DNA (cDNA) molecules within the first plurality of nuclei, wherein reverse transcribing the RNA molecules comprises coupling primers to the RNA molecules, wherein the primers comprise at least one of a poly(T) sequence or a random sequence; (c) dividing the first plurality of nuclei comprising cDNA molecules into at least two primary aliquots, the at least two primary aliquots comprising a first primary aliquot and a second primary aliquot; (d) providing primary nucleic acid tags to the at least two primary aliquots, wherein the primary nucleic acid tags provided to the first primary aliquot are different from the primary nucleic acid tags provided to the second primary aliquot; (e) coupling the cDNA molecules within each of the at least two primary aliquots with the provided primary nucleic acid tags; (f) combining the at least two primary aliquots; (g) dividing the combined primary aliquots into at least two secondary aliquots, the at least two secondary aliquots comprising a first secondary aliquot and a second secondary aliquot; (h) providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tags provided to the first secondary aliquot are different from the secondary nucleic acid tags provided to the second secondary aliquot; (i) coupling the cDNA molecules within each of the at least two secondary aliquots with the provided secondary nucleic acid tags; (j) combining the at least two secondary aliquots; (k) lysing the first plurality of nuclei to release the cDNA molecules from within the first plurality of nuclei to form a lysate; and (l) adding a binding agent to the lysate such that the cDNA molecules bind the binding agent.
  2. The method of claim 1 wherein before step (k), the method further comprises dividing the combined at least two secondary aliquots into at least two final aliquots, the at least two final aliquots comprising a first final aliquot and a second final aliquot.
  3. The method of any one of claims 1-2, further comprising: (m) conducting a template switch of the cDNA molecules bound to the binding agent; and (n) amplifying the cDNA molecules to form an amplified cDNA molecule solution.
  4. The method of claim 3, further comprising: (o) introducing a solid phase reversible immobilization (SPRI) bead solution to the amplified cDNA molecule solution, wherein the ratio of SPRI bead solution to amplified cDNA molecule solution is between 0.9:1 and 0.7:1
  5. The method of any one of claims 1-4, wherein a common adapter sequence is added to the 3'-end of the released cDNA molecules by template switching.
  6. The method of any one of claims 1-5, wherein the primers of step (b) further comprise a first specific barcode; the method further comprising: (p) reverse transcribing RNA molecules within a second plurality of nuclei to form cDNA molecules within the second plurality of nuclei, wherein reverse transcribing the RNA molecules comprises coupling specific primers to the RNA molecules, wherein the primers comprise a second specific barcode and at least one of a poly(T) sequence or a random sequence, wherein the first specific barcode is different from the second specific barcode such that the cDNA molecules from the first plurality of nuclei can be identified in comparison to the cDNA molecules from the second plurality of nuclei; (q) dividing the second plurality of nuclei comprising cDNA molecules into at least two primary aliquots, the at least two primary aliquots comprising a first primary aliquot and a second primary aliquot; (r) providing primary nucleic acid tags to the at least two primary aliquots, wherein the primary nucleic acid tags provided to the first primary aliquot are different from the primary nucleic acid tags provided to the second primary aliquot; (s) coupling the cDNA molecules within each of the at least two primary aliquots with the provided primary nucleic acid tags; (t) combining the at least two primary aliquots; (u) dividing the combined primary aliquots into at least two secondary aliquots, the at least two secondary aliquots comprising a first secondary aliquot and a second secondary aliquot; (v) providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tags provided to the first secondary aliquot are different from the secondary nucleic acid tags provided to the second secondary aliquot; and (w) coupling the cDNA molecules within each of the at least two secondary aliquots with the provided secondary nucleic acid tags.
  7. The method of any one of claims 1-6, wherein the primers each comprise a 5' overhang comprising a 5' overhang sequence located 5' of the poly(T) sequence or the random sequence and wherein each of the nucleic acid tags comprises: a first strand comprising: a barcode sequence comprising a 3' end and a 5' end; and a 3' hybridization sequence and a 5' hybridization sequence flanking the 3' end and the 5' end of the barcode sequence, respectively; and a second strand comprising: a first portion complementary to the 5' overhang sequence of a reverse transcription primer or the 5' hybridization sequence of a previously coupled nucleic acid tag; and a second portion complementary to the 3' hybridization sequence.
  8. The method of any one of claims 1-7, further comprising: ligating at least two of the nucleic acid tags that are bound to the cDNA molecules, wherein the ligation is performed within the first plurality of nuclei.
  9. The method of any one of claims 1-8, further comprising: ligating at least two of the nucleic acid tags that are bound to the released cDNA molecules.
  10. The method of any one of claims 1-9, wherein f the nucleic acid tags comprise a 5' biotin, and wherein the binding agent comprises streptavidin-coated magnetic beads.
  11. The method of any one of claims 1-10, wherein the poly(T) sequence comprises 15 consecutive dTs.
  12. The method of any one of claims 1-11, wherein the random sequence is a random hexamer.
  13. The method of any one of claims 3-12, further comprising sequencing the cDNA molecules amplified in step (n).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of United States Provisional Application No. 62/561,806, filed September 22, 2017, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD The present disclosure relates generally to methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue. The present disclosure also relates to kits for uniquely labeling molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue. In particular, the methods and kits may relate to the labeling of RNAs and/or cDNAs. BACKGROUND Next Generation Sequencing (NGS) can be used to identify and/or quantify individual transcripts from a sample of cells. However, such techniques may be too complicated to perform on individual cells in large samples. In such methods, RNA transcripts are generally purified from lysed cells (i.e., cells that have been broken apart), followed by conversion of the RNA transcripts into complementary DNA (cDNA) using reverse transcription. The cDNA sequences can then be sequenced using NGS. In such a procedure, all of the cDNA sequences are mixed together before sequencing, such that RNA expression is measured for a whole sample and individual sequences cannot be linked back to an individual cell. Methods for uniquely labeling or barcoding transcripts from individual cells can involve the manual separation of individual cells into separate reaction vessels and can require specialized equipment. An alternative approach to sequencing individual transcripts in cells is to use microscopy to identify individual fluorescent bases. However, this technique can be difficult to implement and limited to sequencing a low number of cells. BRIEF DESCRIPTION OF THE DRAWINGS The embodiments disclosed herein will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. FIG. 1 depicts ligation of nucleic acid tags to form a label or barcode.FIG. 2 is a schematic representation of the formation of cDNA by in situ reverse transcription. Panel A depicts a cell that is fixed and permeabilized. Panel B depicts addition of a poly(T) primer, which can template the reverse transcription of polyadenylated transcripts. Panel C depicts addition of a random hexamer, which can template the reverse transcription of substantially any transcript. Panel D depicts the addition of a primer that is designed to target a specific transcript such that only a subset of transcripts may be amplified. Panel E depicts the cell of Panel A after reverse transcription, illustrating a cDNA hybridized to an RNA.FIG. 3A depicts non-templated ligation of a single-stranded adapter to an RNA fragment.FIG. 3B depicts ligation of a single-stranded adapter using a partial duplex with random hexamer primers.FIG. 4 depicts primer binding.FIG. 5 depicts primer binding followed by reverse transcription.FIG. 6 depicts DNA-tagged antibodies for use in labeling cellular proteins.FIG. 7 depicts aptamers for use in labeling cellular proteins.FIG. 8 is a schematic representation of the dividing, tagging, and pooling of cells, according to an embodiment of the present disclosure. As depicted, cells can be divided between a plurality of reaction vessels. One cell is highlighted to show its path through the illustrated process.FIG. 9A depicts an exemplary workflow, according to an embodiment of the present disclosure.FIG. 9B depicts an exemplary workflow, according to another embodiment of the present disclosure.FIG. 10 depicts a reverse transcription primer (BC_0055), according to an embodiment of the present disclosure.FIG. 11 depicts an annealed, first-round barcode oligo, according to an embodiment of the present disclosure.FIG. 12 depicts an annealed, second-round barcode oligo, according to an embodiment of the present disclosure.FIG. 13 depicts an annealed, third-round barcode oligo, according to an embodiment of the present disclosure.FIG. 14 depicts ligation stop oligos, according to an embodiment of the present disclosure.FIG. 15 depicts a single-stranded DNA adapter oligo (BC_0047) ligated to the 3' end of a cDNA, according to an embodiment of the present disclosure.FIG. 16 depicts a PCR product formed using primers BC_0051 and BC_0062 and the 3' adapter oligo (BC_0047) after it has been ligated to barcoded cDNA.FIG. 17 depicts BC_0027, which includes the flow cell binding sequence and the binding site for the TRUSEQ™ read 1 primer and BC_0063, which includes the flow cell binding sequence and the TruSeq multiplex read 2 and index binding sequence. FIG. 17 also illustrates a region for a sample index, which is GATCTG in this embodiment.FIG. 18 is a scatter plot, wherein for each unique barcode combination the number of reads aligning to the human genome (x-axis) and the mouse genome (y-axis) are plotted.FIG. 19 illustrates the size o