Multiplex molecular labeling by sequential hybridization barcoding

Abstract

The present application relates to molecular multiple labeling by sequential hybridization barcoding. Among other things, the present application provides techniques for detecting and / or quantifying nucleic acids in cells, tissues, organs, or organisms. In some embodiments, the present application provides methods for high-throughput profiling of a large number of targets, such as transcripts and / or DNA loci, by sequential barcoding.

Description

[0001] This application is a divisional application of an application with an application date of April 30, 2014, application number 201480037597.5, and invention name “Molecular multiple labeling by sequential hybridization barcoding”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application Serial No. 61 / 817,651, filed April 30, 2013, and U.S. Provisional Application Serial No. 61 / 971,974, filed March 28, 2014, each of which is incorporated herein by reference in its entirety. Technical Field

[0004] The present application relates to, but is not limited to, molecular multiplexing labeling by barcoding via sequential hybridization. Background of the Invention

[0005] Transcriptional profiling of cells is essential for many purposes. Microscopic imaging that can resolve multiple mRNAs in single cells can provide valuable information about transcript abundance and localization, which is important for understanding the molecular basis of cell identification and developing disease treatments. Therefore, there is a need for new and improved methods for profiling transcripts in cells by, for example, microscopic imaging. SUMMARY OF THE INVENTION

[0006] The present invention provides certain insights into challenges or shortcomings associated with prior art techniques for profiling transcripts or DNA loci in cells, particularly in single cells. In addition, the present invention provides new techniques for efficiently performing such profiling, including such profiling of single cells. The provided techniques are broadly useful, including, for example, profiling of isolated cells, cells in tissues, cells in organs, and / or cells in organisms.

[0007] For example, the present invention provides the following insights: existing technologies such as single cell RNA-seq or qPCR require the isolation of single cells and their placement into a multi-well format, which can be a multi-step process that is costly, labor-intensive, and prone to artifacts. In addition, the present invention recognizes that existing in situ sequencing technologies that first use an enzymatic reaction to convert mRNA into a DNA template can be very inefficient (e.g., during the conversion of mRNA to DNA), so that, typically, only a small fraction of the RNA is converted and detected. The present invention provides such specific insights: A major disadvantage of this inefficiency (estimated at 1% for RT and 10% for PLA) is that it can introduce significant noise and (ad) bias in gene expression measurements. The present invention also recognizes that existing spectral mRNA barcoding technologies using single molecule fluorescence in situ hybridization (smFISH) require different fluorescent groups for scale-up and may be limited in the number of barcodes that can be generated. smFISH also requires the probes to be split into barcoded subsets during hybridization. Because smFISH typically uses two or more colors for targets, it produces a high density of objects in the image, which can increase the complexity of data analysis.

[0008] Among other aspects, the present invention provides new techniques for profiling, e.g., transcripts and / or DNA loci, that overcome one or more or all of the problems associated with prior methods of the present invention. In some embodiments, the present invention provides methods for detecting multiple targets (e.g., transcripts or DNA loci) in a cell by a sequential barcoding scheme that allows multiplexing of different targets.

[0009] In some embodiments, the present invention provides a method comprising the steps of:

[0010] (a) performing a first contacting step, the first contacting step comprising contacting a cell comprising a plurality of nucleic acids with a first plurality of detectably labeled oligonucleotides, each of the first plurality of detectably labeled oligonucleotides targeting a nucleic acid and being labeled with a detectable moiety, whereby the composition comprises at least:

[0011] (i) a first oligonucleotide that targets a first nucleic acid and is labeled with a first detectable moiety; and

[0012] (ii) a second oligonucleotide that targets a second nucleic acid and is labeled with a second detectable moiety;

[0013] (b) imaging the cell after the first contacting step to detect the interaction of the first plurality of oligonucleotides with their targets;

[0014] (c) performing a second contacting step, said second contacting step comprising contacting said cell with a second plurality of detectably labeled oligonucleotides, said second plurality comprising oligonucleotides targeting overlapping nucleic acids targeted by said first plurality, such that said second plurality comprises at least:

[0015] (i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, the third oligonucleotide targeting the first nucleic acid; and

[0016] (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, which is targeted to the second nucleic acid,

[0017] wherein said second plurality differs from said first plurality in that at least one of the oligonucleotides present in said second plurality is labeled with a detectable moiety that is different from a corresponding oligonucleotide in said first plurality that targets the same nucleic acid, such that in said second plurality:

[0018] (iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, or the third detectable moiety; and

[0019] (iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety or the fourth detectable moiety,

[0020] wherein the third oligonucleotide is labeled with a different detectable moiety than that used for the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than that used for the second oligonucleotide, or both;

[0021] (d) imaging the cell after said second contacting step to detect the interaction of said second plurality of oligonucleotides with their targets; and

[0022] (e) optionally repeating the contacting and imaging steps, each time using a new plurality of detectably labeled oligonucleotides, wherein the new plurality of detectably labeled oligonucleotides comprises oligonucleotides that target overlapping nucleic acids targeted by the first plurality and the second plurality, wherein each utilized plurality is different from each other utilized plurality due to at least one difference in the detectable portion of the labeling of the oligonucleotides targeting the same nucleic acid.

[0023] In some embodiments, the present invention (e.g., as in Figure 1 ), a method is provided, the method comprising the following steps:

[0024] (a) performing a first contacting step, the first contacting step comprising contacting a cell comprising a plurality of transcripts and a DNA locus with a first plurality of detectably labeled oligonucleotides, each of which is targeted to a transcript or a DNA locus and is labeled with a detectable moiety, whereby the composition comprises at least:

[0025] (i) a first oligonucleotide that targets a first transcript or DNA locus and is labeled with a first detectable moiety; and

[0026] (ii) a second oligonucleotide targeted to a second transcript or DNA locus and labeled with a second detectable moiety;

[0027] (b) imaging the cell after the first contacting step to detect recognition of the first plurality of oligonucleotides by their targets;

[0028] (c) performing a second contacting step, said second contacting step comprising contacting said cell with a second plurality of detectably labeled oligonucleotides, said second plurality comprising oligonucleotides targeted to overlapping transcripts and / or DNA loci targeted by the first plurality, such that said second plurality comprises at least:

[0029] (i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, which is targeted to the first transcript or DNA locus; and

[0030] (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeted to the second transcript or DNA locus, wherein the second plurality differs from the first plurality in that at least one of the oligonucleotides present in the second plurality is labeled with a detectable moiety that is different from the corresponding oligonucleotide in the first plurality that targets the same transcript or DNA locus, such that in the second plurality:

[0031] (iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, or the third detectable moiety; and

[0032] (iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety or the fourth detectable moiety,

[0033] wherein the third oligonucleotide is labeled with a different detectable moiety than that used for the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than that used for the second oligonucleotide, or both;

[0034] (d) imaging the cell after said second contacting step to detect recognition of said second plurality of oligonucleotides by their targets; and

[0035] (e) optionally repeating the contacting and imaging steps, each time using a new plurality of detectably labeled oligonucleotides, the new plurality of detectably labeled oligonucleotides comprising oligonucleotides that target overlapping transcripts or DNA loci targeted by the first plurality and the second plurality, wherein each plurality utilized differs from every other plurality utilized due to at least one difference in the detectable portion of the labeling of the oligonucleotides that target the same transcript or DNA locus.

[0036] In some embodiments, the nucleic acid targeted by the detectably labeled oligonucleotide is a transcript and / or a DNA locus, or comprises a transcript and / or a DNA locus. In some embodiments, the nucleic acid targeted by the detectably labeled oligonucleotide is a transcript, or comprises a transcript. In some embodiments, the nucleic acid targeted by the detectably labeled oligonucleotide is a transcript. In some embodiments, the nucleic acid targeted by the detectably labeled oligonucleotide is a DNA locus, or comprises a DNA locus. In some embodiments, the nucleic acid targeted by the detectably labeled oligonucleotide is a DNA locus. In some embodiments, each plurality of the multiple detectably labeled oligonucleotides used in the contacting step targets the same transcript and / or DNA locus.

[0037] In some embodiments, the multiple detectably labeled oligonucleotides used in the contacting step are referred to as a group of detectably labeled oligonucleotides. In some embodiments, the target of a group of detectably labeled oligonucleotides is referred to as a group of targets. In some embodiments, the target in the group is or includes a transcript. In some embodiments, the target in the group is a transcript. In some embodiments, each target in the group is or includes a transcript. In some embodiments, each target in the group is a transcript. In some embodiments, the target in the group is or includes a DNA locus. In some embodiments, the target in the group is a DNA locus. In some embodiments, each target in the group is or includes a DNA locus. In some embodiments, each target in the group is a DNA locus.

[0038] In some embodiments, the method provided optionally includes the step of removing multiple detectably labeled oligonucleotides after the imaging step. In some embodiments, the method provided includes the step of removing multiple detectably labeled oligonucleotides after each imaging step. In some embodiments, the step of removing includes contacting multiple detectably labeled oligonucleotides with an enzyme that digests the detectably labeled oligonucleotides. In some embodiments, the removal step includes contacting multiple detectably labeled oligonucleotides with a DNA enzyme. In some embodiments, the removal step includes contacting multiple detectably labeled oligonucleotides with an RNA enzyme. In some embodiments, the removal step includes photobleaching.

[0039] In some embodiments, each group includes two or more detectably labeled oligonucleotides targeting the same transcript and / or DNA locus. In some embodiments, two or more detectably labeled oligonucleotides in the group targeting the same transcript and / or DNA locus produce the same detectable signal. In some embodiments, all detectably labeled oligonucleotides in the group targeting the same transcript and / or DNA locus produce the same detectable signal. In some embodiments, wherein the detectably labeled oligonucleotides are labeled with a fluorophore, the detectable signal is a color. In some embodiments, all detectably labeled oligonucleotides in the group targeting the same transcript and / or DNA locus are labeled with a fluorophore that provides the same detectable color.

[0040] In some embodiments, two or more detectably labeled oligonucleotides in a group targeting the same transcript and / or DNA locus have the same detectable label. In some embodiments, all detectably labeled oligonucleotides in a group targeting the same transcript and / or DNA locus have the same detectable label. In some embodiments, all detectably labeled oligonucleotides targeting the same transcript and / or DNA locus have the same fluorophore.

[0041] In some embodiments, the invention provides compositions useful for performing provided methods.

[0042] In some embodiments, the present invention provides a composition comprising a plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid and is labeled with a detectable moiety, such that the composition comprises at least:

[0043] (i) a first oligonucleotide that targets a first nucleic acid and is labeled with a first detectable moiety; and

[0044] (ii) a second oligonucleotide targeted to a second nucleic acid and labeled with a second detectable moiety.

[0045] In some embodiments, the present invention provides a kit comprising a plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid and is labeled with a detectable moiety, such that the kit comprises at least:

[0046] (i) a first oligonucleotide that targets a first nucleic acid and is labeled with a first detectable moiety;

[0047] (ii) a second oligonucleotide targeted to a second nucleic acid and labeled with a second detectable moiety.

[0048] (iii) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeted to the first nucleic acid and labeled with the first detectable moiety, the second detectable moiety, or a third detectable moiety; and

[0049] (iv) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeting nucleic acid and labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety, or the fourth detectable moiety,

[0050] wherein the third oligonucleotide is labeled with a different detectable moiety than the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than the second oligonucleotide, or both.

[0051] In some embodiments, the detectable moiety is or includes a fluorophore.

[0052] In some embodiments, a plurality of detectably labeled oligonucleotides target two or more nucleic acids ("targets"). In some embodiments, the target is or includes a transcript. In some embodiments, the target is a transcript. In some embodiments, the target is RNA. In some embodiments, the target is mRNA. In some embodiments, the target is tRNA. In some embodiments, the target is rRNA. In some embodiments, the target is non-coding RNA. In some embodiments, the target is or includes a DNA locus. In some embodiments, the transcript is a DNA locus. In some embodiments, the target is the locus of a transcript. In some embodiments, different transcripts of a DNA sequence, such as splice variants of a gene, constitute different targets, wherein one or more of the variants can be independently targeted and detected or quantified. In some embodiments, the present invention provides methods and kits for detecting individual splice variants. In some embodiments, the present invention provides methods and kits for detecting single nucleotide polymorphisms (SNPs).

[0053] In some embodiments, provided methods quantify a target, eg, a transcript or a DNA locus.

[0054] In some embodiments, oligonucleotides directed to the same target have the same set of sequences, ie, when they are used in different steps, the differences between the oligonucleotides are in the moieties, not in the sequences.

[0055] Specifically, this application provides the following:

[0056] Item 1. A composition comprising a plurality of detectably labeled oligonucleotides, each of which targets a transcript or a DNA locus and is labeled with a detectable moiety, such that the composition comprises at least:

[0057] (i) a first oligonucleotide targeted to a first transcript or DNA locus and labeled with a first detectable moiety; and

[0058] (ii) a second oligonucleotide targeted to a second transcript or DNA locus and labeled with a second detectable moiety.

[0059] Item 2. A kit comprising a plurality of detectably labeled oligonucleotides, each of which targets a transcript or a DNA locus and is labeled with a detectable moiety, such that the kit comprises at least:

[0060] (i) a first oligonucleotide targeted to a first transcript or DNA locus and labeled with a first detectable moiety;

[0061] (ii) a second oligonucleotide targeted to a second transcript or DNA locus and labeled with a second detectable moiety;

[0062] (iii) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeted to the first transcript or DNA locus and labeled with the first detectable moiety, the second detectable moiety, or a third detectable moiety; and

[0063] (iv) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeted to the second transcript or DNA locus and labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety, or the fourth detectable moiety,

[0064] wherein the third oligonucleotide is labeled with a detectable moiety different from that of the first oligonucleotide or the fourth oligonucleotide is labeled with a detectable moiety different from that of the second oligonucleotide, or both.

[0065] Item 3. The composition or kit as described in any of the preceding items, wherein each oligonucleotide is labeled with a fluorophore.

[0066] Item 4. A composition or kit as described in any of the preceding items, wherein the first oligonucleotide and the second oligonucleotide target different transcripts or DNA loci.

[0067] Item 5. The composition or kit of any one of the preceding items, wherein the first oligonucleotide and the second oligonucleotide target the same transcript or DNA locus.

[0068] Item 6. A composition or kit as described in Item 5, wherein the plurality of detectably labeled oligonucleotides comprises two or more detectably labeled oligonucleotides targeting the same transcript or DNA locus.

[0069] Item 7. The composition or kit as described in Item 5, wherein the first oligonucleotide and the second oligonucleotide have different oligonucleotide sequences.

[0070] Item 8. The composition or kit of any of the preceding items, wherein the first detectable moiety and the second detectable moiety are different.

[0071] Item 9. The composition or kit of any of the preceding items, wherein the first detectable moiety and the second detectable moiety are the same.

[0072] Item 10. The composition or kit of any one of the preceding items, wherein the first oligonucleotide and the second oligonucleotide have a sequence identity of less than 68%.

[0073] Item 11. The composition or kit of any of the preceding items, wherein each oligonucleotide has less than 50% sequence identity with any other oligonucleotide.

[0074] Item 12. The composition or kit of any of the preceding items, wherein each oligonucleotide is less than 29 base pairs in length.

[0075] Item 13. A composition or kit as described in any of the preceding items, wherein the plurality of detectably labeled oligonucleotides targets at least 10 different transcripts and / or DNA loci.

[0076] Item 14. The composition or kit as described in any of the preceding items, comprising at least two detectable moieties.

[0077] Item 15. The composition according to any one of the preceding items, further comprising:

[0078] (iii) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, the third oligonucleotide targeting the first transcript or DNA locus; and

[0079] (iv) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, the fourth oligonucleotide targeting the second transcript or DNA locus,

[0080] wherein the third oligonucleotide is labeled with a different detectable moiety than the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than the second oligonucleotide, or both.

[0081] Item 16. The composition or kit as described in any of the preceding items, wherein the third oligonucleotide is identical in sequence to the first oligonucleotide.

[0082] Item 17. The composition or kit of any of the preceding items, wherein the third oligonucleotide is labeled with a detectable moiety different from that of the first oligonucleotide.

[0083] Item 18. The composition or kit of any one of the preceding items, wherein the fourth oligonucleotide is identical in sequence to the second oligonucleotide.

[0084] Item 19. The composition or kit of any of the preceding items, wherein the fourth oligonucleotide is labeled with a detectable moiety different from that of the second oligonucleotide.

[0085] Item 20. A method, comprising the following steps:

[0086] (a) performing a first contacting step, the first contacting step comprising contacting a cell comprising a plurality of nucleic acids with a first plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid and is labeled with a detectable moiety, such that the composition comprises at least:

[0087] (i) a first oligonucleotide that targets a first nucleic acid and is labeled with a first detectable moiety; and

[0088] (ii) a second oligonucleotide targeted to a second nucleic acid and labeled with a second detectable moiety;

[0089] (b) imaging the cell after the first contacting step to detect the interaction of the first plurality of oligonucleotides with their targets;

[0090] (c) performing a second contacting step, said second contacting step comprising contacting said cell with a second plurality of detectably labeled oligonucleotides, said second plurality comprising oligonucleotides targeting overlapping nucleic acids targeted by said first plurality, such that said second plurality comprises at least:

[0091] (i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, the third oligonucleotide targeting the first nucleic acid; and

[0092] (ii) a fourth oligonucleotide, said fourth oligonucleotide being optionally identical in sequence to said second oligonucleotide, said third oligonucleotide being targeted to said second oligonucleotide,

[0093] wherein said second plurality differs from said first plurality in that at least one of the oligonucleotides present in said second plurality is labeled with a detectable moiety that is different from a corresponding oligonucleotide in said first plurality that targets the same nucleic acid, such that in said second plurality:

[0094] (iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, or the third detectable moiety; and

[0095] (iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety or the fourth detectable moiety,

[0096] wherein the third oligonucleotide is labeled with a different detectable moiety than that used for the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than that used for the second oligonucleotide, or both;

[0097] (d) imaging said cell after said second contacting step, thereby to detect the interaction of said second plurality of oligonucleotides with their targets; and

[0098] (e) optionally repeating the contacting step and the imaging step, each time using a new plurality of detectably labeled oligonucleotides, wherein the new plurality of detectably labeled oligonucleotides comprises oligonucleotides that target overlapping nucleic acids targeted by the first plurality and the second plurality, wherein each plurality utilized is different from every other plurality utilized due to at least one difference in the detectable portion of the labeling of the oligonucleotides that target the same nucleic acid.

[0099] Item 21. A method, comprising the following steps:

[0100] (a) performing a first contacting step, the first contacting step comprising contacting a cell comprising a plurality of transcripts and a DNA locus with a first plurality of detectably labeled oligonucleotides, each of the first plurality of detectably labeled oligonucleotides being targeted to a transcript or a DNA locus and being labeled with a detectable moiety, whereby the composition comprises at least:

[0101] (i) a first oligonucleotide targeted to a first transcript or DNA locus and labeled with a first detectable moiety; and

[0102] (ii) a second oligonucleotide targeted to a second transcript or DNA locus and labeled with a second detectable moiety;

[0103] (b) imaging the cell after the first contacting step to detect hybridization of the first plurality of oligonucleotides to their targets;

[0104] (c) performing a second contacting step, said second contacting step comprising contacting said cell with a second plurality of detectably labeled oligonucleotides, said second plurality comprising oligonucleotides targeted to overlapping transcripts and / or DNA loci targeted by said first plurality, such that said second plurality comprises at least:

[0105] (i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, the third oligonucleotide targeting the first transcript or DNA locus; and

[0106] (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, the fourth oligonucleotide targeting the second transcript or DNA locus,

[0107] wherein said second plurality differs from said first plurality in that at least one of the oligonucleotides present in said second plurality is labeled with a detectable moiety that is different from a corresponding oligonucleotide in said first plurality that targets the same transcript or DNA locus, such that in said second plurality:

[0108] (iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, or the third detectable moiety; and

[0109] (iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety or the fourth detectable moiety,

[0110] wherein the third oligonucleotide is labeled with a different detectable moiety than that used for the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than that used for the second oligonucleotide, or both;

[0111] (d) imaging the cell after the second contacting step to detect hybridization of the second plurality of oligonucleotides to their targets; and

[0112] (e) optionally repeating the contacting step and the imaging step, each time using a new plurality of detectably labeled oligonucleotides, the new plurality of detectably labeled oligonucleotides comprising oligonucleotides targeting overlapping transcripts or DNA loci targeted by the first plurality and the second plurality, wherein each plurality utilized is different from every other plurality utilized due to at least one difference in the detectable portion of the labeling of the oligonucleotides targeting the same transcript or DNA locus.

[0113] Item 22. A method as described in Item 20 or 21, wherein each of the multiple detectably labeled oligonucleotides comprises F detectable moieties, wherein F is at least 2.

[0114] Item 23. A method as described in any one of Items 20-22, comprising N contacting steps, wherein N is at least 2.

[0115] Item 24. A method as described in any one of Items 20-23, wherein each of the multiple targets (F) of the detectably labeled nucleotides N transcripts and / or DNA loci.

[0116] Item 25. A method as described in any of Items 20-23, wherein each of the multiple targets of the detectably labeled oligonucleotides is less than (F) N transcripts and / or DNA loci.

[0117] Item 26. A method as described in any of Items 20-25, wherein each plurality of detectably labeled oligonucleotides targets the same transcript and / or DNA locus.

[0118] Item 27. A method as described in any of Items 20-26, which optionally includes a step of removing multiple detectably labeled oligonucleotides after the imaging step.

[0119] Item 28. A method as described in any of Items 20-27, comprising a step of removing multiple detectably labeled oligonucleotides after each imaging step.

[0120] Item 29. A method as described in Item 28, wherein the removing step comprises contacting the plurality of detectably labeled oligonucleotides with an enzyme that digests the detectably labeled oligonucleotides.

[0121] Item 30. A method as described in Item 29, wherein the removing step comprises contacting the plurality of detectably labeled oligonucleotides with a DNase.

[0122] Item 31. A method as described in Item 29, wherein the removing step comprises contacting the plurality of detectably labeled oligonucleotides with an RNase.

[0123] Item 32. A method as described in any of Items 20-31, wherein the removing step includes photobleaching.

[0124] Item 33. A method as described in any of Items 20-32, wherein each plurality comprises two or more detectably labeled oligonucleotides targeting the same transcript or DNA locus.

[0125] Item 34. A method as described in Item 33, wherein all detectably labeled oligonucleotides targeting the same transcript or DNA locus are labeled with a fluorophore of the same color.

[0126] Item 35. A method as described in Item 33, wherein all detectably labeled oligonucleotides targeting the same transcript or DNA locus are labeled with the same fluorophore.

[0127] Item 36. A method as described in any of Items 20-35, wherein each plurality of detectably labeled oligonucleotides is different from another one.

[0128] Item 37. A method as described in any of Items 20-36, wherein the third oligonucleotide is labeled with a detectable portion different from that of the first oligonucleotide, and the fourth oligonucleotide is labeled with the same detectable portion as that of the second oligonucleotide; or the third oligonucleotide is labeled with the same detectable portion as that of the first oligonucleotide, and the fourth oligonucleotide is labeled with a detectable portion different from that of the second oligonucleotide.

[0129] Item 38. A method as described in any of Items 20-36, wherein the third oligonucleotide is labeled with a detectable moiety different from that of the first oligonucleotide, and the fourth oligonucleotide is labeled with a detectable moiety different from that of the second oligonucleotide.

[0130] Item 39. A method as described in any of Items 20-38, wherein the imaging step includes imaging the cell after the contacting step, so that hybridization of multiple detectably labeled oligonucleotides to their targets is quantified.

[0131] Item 40. The method of any one of Items 20-39, further comprising HCR, light sheet microscopy, CLARITY, or a combination thereof.

[0132] Item 41. The composition, kit or method of any of the preceding items, wherein the targets of each plurality of detectably labeled oligonucleotides are predetermined.

[0133] Item 42. The composition, kit or method of any of the preceding items, wherein the amount of each detectably labeled oligonucleotide is predetermined.

[0134] Item 43. The composition, kit or method of any of the preceding items, wherein the detectably labeled oligonucleotide is hybridized to an intermediate oligonucleotide that hybridizes to the target.

[0135] Item 44. A composition, kit or method as described in Item 43, wherein the intermediate oligonucleotide comprises a sequence complementary to the sequence of the target and an overhang sequence complementary to the detectably labeled oligonucleotide.

[0136] Item 45. The composition, kit or method of any of the preceding items, wherein there are two or more intermediate oligonucleotides hybridized to the target.

[0137] Item 46. A composition as described in Item 45, wherein each intermediate oligonucleotide that hybridizes to the target comprises the same overhang sequence.

[0138] Item 47. The composition, kit or method of any of the preceding items, wherein the detectably labeled oligonucleotide comprises a 5'-phosphorylation.

[0139] Item 48. The composition, kit or method of any of the preceding items, wherein the detectably labeled oligonucleotide comprises one or more uracils.

[0140] Item 49. The composition, kit or method of any one of Items 43-46, wherein the detectably labeled oligonucleotide has a recessed 3'-end after hybridization to the intermediate oligonucleotide.

[0141] Item 50. The method according to any one of Items 20 to 49, further comprising the following steps:

[0142] (f) performing a contacting step comprising contacting the cell comprising the plurality of nucleic acids with a plurality of intermediate oligonucleotides, each of the plurality of intermediate oligonucleotides:

[0143] (i) targets a nucleic acid and is optionally labeled with a detectable moiety; and

[0144] (ii) comprises an overhang sequence after hybridization to the target; and

[0145] (g) optionally imaging the cell to detect the interaction between the intermediary oligonucleotide and its target.

[0146] Item 51. A method as described in Item 50, wherein the intermediate oligonucleotide is not labeled with a detectable moiety.

[0147] Item 52. The method of Item 50, wherein step (g) is not present.

[0148] Item 53. A method as described in Item 50, wherein step (f) and optionally step (g) are performed before step (a).

[0149] Item 54. A method as described in any of Items 20-53, wherein the removal step does not destroy the intermediate oligonucleotide.

[0150] Item 55. A method as described in any of Items 50-54, wherein the nucleic acid is a transcript or a DNA locus.

[0151] Item 56. A method as described in any of Items 20-55, wherein the cell is a human cell.

[0152] Item 57. A method as described in any of Items 20-56, wherein the cell is within a tissue or organ.

[0153] Item 58. The composition, kit or method of any of the preceding items, wherein at least one detectably labeled oligonucleotide is HCR-labeled.

[0154] Item 59. The composition, kit or method of any of the preceding items, wherein at least one detectably labeled oligonucleotide hybridizes to an intermediate oligonucleotide that hybridizes to a transcript or a DNA locus.

[0155] Item 60. A method as described in any of the preceding items, comprising a removal step, wherein the removal step comprises using exonuclease III, Lambda exonuclease or a uracil-specific excision reagent.

[0156] Item 61. A light sheet illumination microscope in which the sample is illuminated from the side.

[0157] Item 62. The microscope of Item 61, wherein the sample is illuminated from its side and the light sheet is parallel to the sample stage.

[0158] Item 63. A microscope as described in Item 61 or 62, wherein the light sheet is perpendicular to the detection object.

[0159] Item 64. A microscope as described in any of Items 61-63, wherein the sample is scanned along the z-axis.

[0160] Item 65. A method for preparing a target nucleic acid, wherein the target nucleic acid comprises a first sequence, the method comprising the following steps:

[0161] 1) providing a first nucleic acid comprising the first sequence, wherein the first sequence is flanked at both ends by nicking endonuclease sites;

[0162] 2) amplifying the first nucleic acid or a portion of the first nucleic acid to provide a second nucleic acid, the second nucleic acid comprising the first sequence and flanking nicking endonuclease sites; and

[0163] 3) contacting the second nucleic acid with one or more nicking endonucleases corresponding to the flanking nicking endonuclease sites.

[0164] Item 66. A method as described in Item 65, wherein the two flanking nicking endonuclease sites are the same.

[0165] Item 67. A method as described in Item 66, wherein a nicking endonuclease corresponding to the same nicking endonuclease site is used.

[0166] Item 68. A method as described in Item 65, wherein the two flanking nicking endonuclease sites are different.

[0167] Item 69. A method as described in Item 68, wherein two nicking endonucleases are used, each of the two nicking endonucleases independently corresponding to a nicking endonuclease site.

[0168] Item 70. A method as described in any one of Items 65-69, which includes a denaturation step, wherein the double-stranded nucleic acid is converted into a single-stranded nucleic acid.

[0169] Item 71. A method for preparing a target nucleic acid, wherein the target nucleic acid comprises a first sequence, the method comprising the following steps:

[0170] 1) providing a first nucleic acid comprising the first sequence or its complementary sequence, wherein the first sequence or its complementary sequence is flanked by at least one restriction site;

[0171] 2) amplifying the first nucleic acid or a portion of the first nucleic acid to provide a second nucleic acid, the second nucleic acid comprising the first sequence and the at least one flanking restriction site; and

[0172] 3) contacting the second nucleic acid with a restriction enzyme corresponding to the at least one flanking restriction site to provide a third nucleic acid comprising a recessed end;

[0173] 4) contacting the third nucleic acid with a nuclease to selectively digest the strand containing the complementary sequence, if present, while retaining the strand containing the first sequence.

[0174] Item 72. A method as described in Item 71, wherein the first sequence or its complementary sequence is independently flanked by restriction sites at both ends.

[0175] Item 73. A method as described in Item 71 or 72, wherein the second nucleic acid is contacted with a restriction enzyme corresponding to each of the at least one flanking restriction site.

[0176] Item 74. A method as described in any of Items 71-73, wherein the chain containing the complementary sequence has a 3' recessed end after restriction digestion.

[0177] Item 75. A method as described in any of Items 71-74, wherein the chain containing the first sequence has a 5' recessed end after restriction digestion.

[0178] Item 76. A method as described in any of Items 71-75, wherein the nuclease is exonuclease III.

[0179] Item 77. A method as described in any of Items 65-76, wherein the target nucleic acid is DNA.

[0180] Item 78. A method as described in any of Items 65-77, wherein the target nucleic acid has a sequence identical to the first sequence.

[0181] Item 79. A method as described in any of Items 65-78, wherein the second nucleic acid and / or the third nucleic acid are optionally and independently modified prior to a subsequent contacting step.

[0182] Item 80. A method as described in any of Items 65-79, wherein the target nucleic acid is an intermediate oligonucleotide comprising a first sequence and a second sequence, wherein the first sequence hybridizes with the target and the second sequence hybridizes with a detectably labeled oligonucleotide labeled with HCR.

[0183] Item 81. A method as described in any of Items 65-79, wherein the target nucleic acid is a bridging probe.

[0184] definition

[0185] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to: mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, animals can be transgenic animals, genetically engineered animals, and / or clones.

[0186] Approximately: As used herein, the term "approximately" or "about" with respect to numbers is generally taken to include numbers that fall within 5%, 10%, 15%, or 20% in either direction (more or less) of the number, unless otherwise stated or otherwise obvious from the context (unless such a number would be less than 0% or more than 100% of the possible value). In some embodiments, the use of the term "about" with respect to dosage means ± 5 mg / kg / day.

[0187] Homology: "Homology" or "identity" or "similarity" refers to the sequence similarity between two nucleic acid molecules. Homology and identity can be determined separately by comparing positions in each sequence that can be aligned for comparison purposes. When equivalent positions in the compared sequences are occupied by the same base, the molecules are identical at that position; when equivalent sites are occupied by the same or similar nucleic acid residues (e.g., similar in steric and / or electronic properties), the molecules can be said to be homologous (similar) at that position. The percentage expressed as homology / similarity or identity refers to the function of the number of identical or similar nucleic acids at the positions shared by the compared sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with the sequences described herein. In the comparison of two sequences, the absence of residues (amino acids or nucleic acids) or the presence of additional residues also reduces identity and homology / similarity.

[0188] In some embodiments, the term "homology" describes a comparison based on mathematical sequence similarity, which is used to identify genes with similar functions or motifs. The nucleic acid sequences described herein can be used as "query sequences" to search against public databases, for example, to identify other family members, related sequences or homologs. In some embodiments, this search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. In some embodiments, a BLAST nucleotide search can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. In some embodiments, in order to obtain a comparison of gaps for comparison purposes, the GappedBLASAT described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402 can be used. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and BLAST) can be used (see www.ncbi.nlm.nih .gov).

[0189] Identity: As used herein, "identity" means the percentage of nucleotide residues at corresponding positions in two or more sequences that are identical when the sequences are aligned for maximum sequence matching, ie, taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Biotechnology, 1996; Math., 48:1073 (1988). Methods for determining identity are designed to give the maximum match between the sequences tested. In addition, methods for determining identity are incorporated into publicly available computer programs. Computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN and FASTA (Altschul, SF et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., J. Mol. Biol. 215:403-410 (1990)).The well-known Smith Waterman algorithm may also be used to determine identity.

[0190] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, eg, in a test tube or reaction vessel, in cell culture, etc., rather than in an organism (eg, an animal, plant, and / or microorganism).

[0191] In vivo: As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant, and / or microorganism).

[0192] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotide monomers, containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate linkages, or modified linkages.

[0193] Oligonucleotides of the present invention can be of various lengths. In specific embodiments, oligonucleotide length can be in the range of from about 2 nucleotides to about 200 nucleotides. In various related embodiments, single-stranded, double-stranded and triple-stranded oligonucleotide length can be in the range of from about 4 nucleotides to about 10 nucleotides, from about 10 nucleotides to about 50 nucleotides, from about 20 nucleotides to about 50 nucleotides, from about 15 nucleotides to about 30 nucleotides, from about 20 nucleotides to about 30 nucleotides. In some embodiments, oligonucleotide length is in the range of from about 9 nucleotides to about 39 nucleotides. In some embodiments, oligonucleotide length is at least 4 nucleotides. In some embodiments, oligonucleotide length is at least 5 nucleotides. In some embodiments, oligonucleotide length is at least 6 nucleotides. In some embodiments, oligonucleotide length is at least 7 nucleotides. In some embodiments, oligonucleotide length is at least 8 nucleotides. In some embodiments, oligonucleotide length is at least 9 nucleotides. In some embodiments, oligonucleotide length is at least 10 nucleotides. In some embodiments, oligonucleotide length is at least 11 nucleotides. In some embodiments, the oligonucleotide length is at least 12 nucleotides. In some embodiments, the oligonucleotide length is at least 15 nucleotides. In some embodiments, the oligonucleotide length is at least 20 nucleotides. In some embodiments, the oligonucleotide length is at least 25 nucleotides. In some embodiments, the oligonucleotide length is at least 30 nucleotides. In some embodiments, the oligonucleotide is a duplex of a complementary chain of at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a duplex of a complementary chain of at least 21 nucleotides in length.

[0194] Predetermined: Predetermined means deliberately chosen, e.g., as opposed to randomly occurring or being achieved. Compositions that may include certain individual oligonucleotides are not "predetermined" compositions: they are individual oligonucleotides that are accidentally produced by a process that cannot be controlled to intentionally produce a particular oligonucleotide. In some embodiments, a predetermined composition is a composition that can be intentionally reproduced (e.g., by repeating a controlled process).

[0195] Sample: As used herein, the term "sample" refers to a biological sample obtained or derived from a source of interest as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample includes a biological tissue or a biological fluid. In some embodiments, the biological sample is or includes bone marrow; blood; blood cells; ascites; tissue or fine needle aspiration biopsy samples; body fluids containing cells; free nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal effusion; pleural effusion; feces; lymph; gynecological fluid; skin swabs; vaginal swabs; oral swabs; nasal swabs; washing or lavage fluids such as catheter lavage fluid or bronchoalveolar lavage fluid; aspirates; scrapings; bone marrow samples; tissue biopsy samples; surgical samples; feces, other body fluids, secretions and / or excretions; and / or cells from which they are derived, etc. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "raw sample" obtained directly from a source of interest by any appropriate means. For example, in some embodiments, the original biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluids (e.g., blood, lymph, feces, etc.). In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing (e.g., by removing one or more components of the original sample and / or by adding one or more reagents to) the original sample. For example, filtering using a semipermeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the original sample to techniques such as amplification or reverse transcription of mRNA, separation and / or purification of certain components, etc.

[0196] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a compound or composition provided by the present invention is administered, for example, for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms, etc.) and plants. In some embodiments, the subject may be suffering from a disease, disorder, and / or condition or be susceptible to a disease, disorder, and / or condition.

[0197] Substantially: As used herein, the term "substantially" refers to a qualitative condition of the total or near-total extent or degree of a characteristic or property of interest. It will be understood by those of ordinary skill in the biological arts that biological and chemical phenomena rarely, if ever, proceed completely and / or to perfection or achieve or avoid absolute results. Thus the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0198] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed as having and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0199] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition is an individual who has a higher risk of developing a disease, disorder, and / or condition compared to members of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with a disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may show symptoms of a disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not show symptoms of a disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop a disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop a disease, disorder, and / or condition.

[0200] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying onset, reducing severity, and / or reducing the occurrence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of a disease, disorder, and / or condition, for the purpose of, for example, reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0201] Wild-type: As used herein, the term "wild-type" has its art-understood meaning and refers to an entity having structure and / or activity as found in nature in a "normal" (as opposed to mutated, diseased, altered, etc.) state or environment. One of ordinary skill in the art will appreciate that wild-type genes and polypeptides typically exist in a variety of different forms (e.g., alleles). BRIEF DESCRIPTION OF THE DRAWINGS

[0202] Figure 1 The methods provided by the present disclosure are presented in Figure 1 middle.

[0203] FIG2 . Exemplary sequential barcoding of the provided methods. (a) Schematic diagram of sequential barcoding. In each round of hybridization, multiple probes (e.g., 24) are hybridized to each transcript, imaged and subsequently stripped by DNase I treatment. The same probe sequence can be used in different rounds of hybridization, but the probes are coupled to different fluorophores. (b) Composite four-color FISH data from three rounds of hybridization on multiple yeast cells. Twelve genes were encoded by two rounds of hybridization, and the third round of hybridization was performed using the same probes as hybridization 1. The boxed area is magnified in the lower right corner of each image. Matching spots are displayed and barcodes are extracted. Non-colocalized spots, not intended to be limited by theory, may be due to nonspecific binding of probes in the cell and mishybridization. The number of each barcode is quantified to provide the abundance of the corresponding transcript in a single cell. (c) Exemplary barcodes. mRNA1: yellow-blue-yellow; mRNA2: green-purple-green; mRNA3: purple-blue-purple; and mRNA4: blue-purple-blue.

[0204] Figure 3 .Schematic diagram of sequential hybridization and barcoding. (a) Schematic diagram of sequential hybridization and barcoding. (b) Schematic diagram of FISH image of cells. In each round of hybridization, the same spot is detected, but the dye associated with the transcript changes. The identity of the mRNA is encoded in the temporal sequence of the hybridized dye.

[0205] Figure 4 DNase I effectively removes smFISH probes bound to mRNA. DNase I effectively removes smFISH probes bound to mRNA. Spots were imaged before and after 4 hours of DNase I treatment in anti-bleach buffer. The mean, median, and STD of the intensity ratios after treatment were 11.5%, 8.3%, and 11%. The ratio of the spot intensity for each spot was plotted before and after DNase I treatment. n=1084 spots.

[0206] Figure 5 .Photobleaching after DNase I treatment to remove residual intensity. Photobleaching after DNase I treatment to remove residual intensity. Spots were bleached by 10 seconds of excitation after 4 hours of DNase I treatment. The mean, median and STD of the intensity ratio after bleaching were 0.03%, 0.01% and 0.049%. The ratio of the spot intensity for each spot was plotted before and after DNase I treatment. n = 1286 spots.

[0207] Figure 6.After multiple rounds of rehybridization, the mRNA is stable. After multiple rounds of rehybridization, the mRNA is stable. The intensity distribution of smRISH spots is plotted after 6 hybridizations. Two hybridizations are repeated 3 times to produce 6 total hybridizations. Spots are identified by colocalization with spots in the next identical hybridization. For each box plot, the number of spots counted is between 191 and 1337.

[0208] Figure 7 Per cell, the percentage of barcodes identified from the first two rounds of hybridization that reappeared in the subsequent round of hybridization. Per cell, the percentage of barcodes identified from the first two rounds of hybridization that reappeared in the subsequent round of hybridization. Barcodes were identified by colocalization across all three hybridizations. 77.9 ± 5.6% of barcodes reappeared. n = 37 cells.

[0209] Figure 8 .Point-wise displacement between FISH spots in hybridization 1 and hybridization 3. Point-wise displacement between FISH spots in hybridization 1 and hybridization 3. FISH spots in Cy5 images in hybridization 1 and hybridization 3 were extracted and fitted with 2D Gaussians. The point-wise displacement is shown in a 3D histogram. The standard deviation is 105.8 nm, indicating that mRNA can be localized to 100 nm between two rounds of hybridization. n = 1199 spots.

[0210] Fig. 9 .Barcodes identified between repeated hybridizations of the same probe set (hybridization 1 and hybridization 3). Barcodes identified between repeated hybridizations of the same probe set (hybridization 1 and hybridization 3). Barcodes identified by colocalization between hybridizations. Each column corresponds to an individual cell. Each row corresponds to a specific barcode identified between hybridization 1 and hybridization 3. Bold row names correspond to repeated color barcodes that should colocalize between hybridization 1 and hybridization 3. Non-bold row names correspond to false positive barcodes. For example, a large number of barcodes were detected for (Alexa 532, Alexa 532), indicating colocalization at the midpoint of the Alexa 532 channel, n=37 cells. Al532=Alexa 532. Al594=Alexa 594. Al647=Alexa 647.

[0211] Fig.10. Single-cell mRNA levels from barcode extraction. Single-cell mRNA levels from barcode extraction. Barcodes were identified by colocalization between hybridization 1 and hybridization 2. Each column corresponds to an individual cell. n = 37 cells. A1532 = Alexa 532. A1594 = Alexa 594. A1647 = Alexa 647. From top to bottom: YLR194c, CMK2, GYP7, PMC1, NPT1, SOK2, UIP3, RCN2, DOA1, HSP30, PUN1, and YPS1.

[0212] Fig.11 DNase I stripping of Nanog Alexa 647 probes in mouse embryonic stem cells (mESCs). DNase I stripping of Nanog Alexa 647 probes in mouse embryonic stem cells (mESCs). Forty-eight probes targeting Nanog in mESCs were hybridized. The probes were stripped by incubation with DNase I at a concentration of 3 units / μL for 30 minutes.

[0213] Fig.12 .Rehybridization of Nanog mRNA in mouse embryonic stem cells (mESC). Rehybridization of Nanog mRNA in mouse embryonic stem cells (mESC). The probe was stripped by incubation with DNase I at a concentration of 3 units / μL for 30 minutes. The Nanog Alexa 647 probe was rehybridized for 12 hours and imaged. The image is a 2D maximum projection generated from a z-stack of 11 images taken every 1.5 μm.

[0214] Fig.13 HCR detection of β-actin (red) in the cortex and visualization with a retrograde tracer (fluorescent gold, green) in 100 μm coronal sections. Whole coronal sections were imaged at both 10X and 60X (magnification inset). In the 60X image, individual red dots correspond to single β-actin mRNA molecules. β-actin expression can be quantified by counting fluorescent foci while simultaneously examining a subset of neurons labeled with a retrograde tracer (green).

[0215] Fig.14 Detectably labeled oligonucleotides labeled with HCR are as specific as smFISH probes directly labeled with fluorophores in detecting single molecules of RNA in 20 μm brain sections. HCR probe (left) and smFISH probe (right) simultaneously target β-actin and colocalize. Note the improved S / N of HCR.

[0216] Fig.15Detectably labeled oligonucleotides labeled with HCR rehybridized well in 20 μm brain sections. HCR spots colocalized in hyb1 and hyb2 with DNase treatment between the two hybridizations. This shows that HCR can be fully integrated into the seqFISH protocol.

[0217] Fig.16 .CLARITY AND NISSl: 1 mm thick coronal sections (bregma AP, 2.3-1.3 mm) of Thy-1-eYFP mouse brains were clarified and stained with NeuroTrace fluorescent Nissl stain (1:100 dilution, 48 hours, RT). Left, 3D coronal rendering of the motor cortex. Right, 100 μm-thick section of layer V motor cortex. Arrows indicate apical dendrites of pyramidal neurons (red - Nissl fluorescence, green - eYFP).

[0218] Fig.17 . Schematic diagram of an exemplary light sheet microscope.

[0219] Fig.18 .SPIM detection of single mRNAs in 100 μm CLARITY brain sections. Sections were scanned across 100 μm. Images were then registered and stitched to a 3D reconstruction. Diffraction-limited spots in the images correspond to single β-actin mRNAs detected by HCR. Scale bar is 15 μm.

[0220] Fig.19 .Connectivity map of the corticalsomatic sensorimoto subnetwork at consecutive coronal levels from the Allen Reference Atlas. It shows that each of the four major components of the somatosensory motor area (SSp, SSs, MOp and MOs) is divided into four functional domains. These functionally related domains are extensively interconnected with all the others and form four major cortical somatosensory motor subnetworks: orofaciopharyngeal (orf, blue), upper limb (ul, green), lower limb and trunk (ll / tr, red) and whiskers (bfd.cm&w, yellow). The numbers indicate the position of the slice relative to bregma (mm). The provided methods can characterize the connectivity and molecular identity of projection neurons in each of these different domains within different subnetworks.

[0221] Fig. 20.Exemplary informatics workflow for automatically detecting and mapping retrogradely labeled neurons and genetic barcode information. A. Raw image labeled with CTb (pink) and Nissl stain (blue). The boxed area shows a close-up of a CTb-labeled neuron. B. The multi-channel raw image is converted to grayscale for segmentation. C. Individual tracer channel images are run through a segmentation pipeline that discretely separates tissue background from labeled cells. White dots are reconstructions of labeled somas. D. The re-integrated multi-view tiff is associated with coronal slices in the ARA for registration. E. Using the provided developed registration software, the multi-view tiff is warped to align both the outline of the tissue and the cytoarchitecture description with its corresponding ARA level. F. Cells extracted by the segmentation process are spatially normalized and can be associated with layer-specific or sub-nucleus-specific ROIs within the ARA. G. The segmented and registered marker reconstructions, as well as their accompanying seqFISH data, are publicly available on the iConnectome FISH viewer. The Analysis tab provides information about the injection site, tracer type, and the number of cells labeled by the ROI, which can further interpret layer-specific cell counts and gene expression of the cells.

[0222] Fig.21 .Hybridization chain reaction (HCR) rehybridization using exonuclease III (Exo III). (a) Schematic representation of exoIII digestion of bridging strands and HCR polymers. ExoIII digests the bridging strands and HCR polymers into dNMPs in the 3' to 5' direction, leaving an intermediate probe strand bound to a target (e.g., mRNA). The new bridging strand can then be hybridized to a target binding probe with a different starting sequence that initiates polymerization of different hairpin sets with different fluorescent dyes. (b) Raw data illustrating the application of the schematic shown in (a) using a probe against β-actin (Actb) transcript in a T3T mouse fibroblast cell line.

[0223] Fig. 22 .Hybridization chain reaction (HCR) rehybridization using Lambda exonuclease (λ-exo). (a) Illustration of λ-exo digestion of bridging strands. λ-exo selectively digests 5' phosphorylated bridging strands in the 5' to 3' direction, releasing HCR polymers from intermediate probes bound to a target (e.g., mRNA). The released polymers are washed away by wash buffer. The new bridging strands can then be hybridized to target binding probes with different starting sequences that initiate polymerization of different hairpin sets with different fluorescent dyes. (b) Raw data illustrating the application of the schematic shown in (a) using a probe against β-actin (Actb) transcripts in a T3T mouse fibroblast cell line.

[0224] Fig.23 .Hybridization chain reaction (HCR) rehybridization using uracil-specific excision reagent (USER). (a) Illustration of USER digestion of a bridge strand. USER selectively digests deoxyuridine nucleotides in the bridge strand, causing the bridge strand to become fragmented. The fragments then dissociate from the intermediate probe strand, releasing HCR polymers from the probe bound to the target (e.g., mRNA). The released polymers are washed away by wash buffer. The new bridge strand can then be hybridized to a target binding probe with a different starting sequence that initiates polymerization of different hairpin sets with different fluorescent dyes. (b) Raw data illustrating the application of the schematic shown in (a) using a probe against β-actin (Actb) transcript in a T3T mouse fibroblast cell line.

[0225] Fig.24 .Exemplary removal of detectably labeled oligonucleotides using complementary oligonucleotides (cTOE).

[0226] Fig.25 .Exemplary oligonucleotide preparation. The original oligonucleotide (probe as demonstrated in this figure) library contains several probe sublibraries. Each sublibrary has a specific set of primers that can be used to amplify the sublibrary using PCR. When the desired sublibrary is amplified, the product is incubated with a nicking enzyme. The enzyme cuts the phosphodiester bond at the recognition site on the probe chain. The resulting product is denatured and run on a denaturing gel, allowing the desired probe sequence to be released. The probe band can then be cut from the gel and extracted. The extracted product can be used for hybridization.

[0227] Fig.26 .Exemplary oligonucleotide preparation. The product is the third band on the gel. The library has many different primers associated with it, one primer set for each subset of probes. Exemplary primers are random 20 nucleotide sequences with a GC content of 45-55% and a Tm of approximately 55°C. The nicking endonuclease site is GTCTCNN; the corresponding nicking endonuclease is Nt.BsmAI. The product probe is a 20-mer DNA sequence complementary to the mRNA of interest with a GC content of 45-55%.

[0228] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0229] The present invention provides, among other things, novel methods, compositions and / or kits for nucleic acid (eg, transcripts and / or DNA loci) profiling in cells.

[0230] In some embodiments, the present invention provides methods for nucleic acid (e.g., transcripts and / or DNA loci) profiling in cells. In some embodiments, the provided methods profile multiple targets in a single cell. The provided methods can, among other things, profile a large number of targets (transcripts, DNA loci, or a combination thereof) with a limited number of detectable labels by sequential barcoding.

[0231] Figure 1 As shown, the present invention provides a method in which multiple rounds of hybridization (contacting step) with labeled probes are used to analyze the nucleic acid (e.g., mRNA) present in the cell. In particular, Figure 1 As shown in , sets of probes that hybridize to nucleic acid targets in cells are provided, wherein the probes within a single set are labeled (i.e., detectably labeled oligonucleotides that hybridize to different targets) and, further, at least one probe in different sets is differently labeled.

[0232] In some embodiments, the present invention (e.g., as in Figure 1 ), a method comprising the following steps is provided:

[0233] (a) performing a first contacting step, the first contacting step comprising contacting a cell comprising a plurality of transcripts and a DNA locus with a first plurality of detectably labeled oligonucleotides, each of the first plurality of detectably labeled oligonucleotides being targeted to a transcript or a DNA locus and being labeled with a detectable moiety, whereby the composition comprises at least:

[0234] (i) a first oligonucleotide that targets a first transcript or DNA locus and is labeled with a first detectable moiety; and

[0235] (ii) a second oligonucleotide targeted to a second transcript or DNA locus and labeled with a second detectable moiety;

[0236] (b) imaging the cell after the first contacting step to detect hybridization of the first plurality of oligonucleotides to their targets;

[0237] (c) performing a second contacting step, said second contacting step comprising contacting said cell with a second plurality of detectably labeled oligonucleotides, said second plurality comprising oligonucleotides targeted to overlapping transcripts and / or DNA loci targeted by said first plurality, such that said second plurality comprises at least:

[0238] (i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, the third oligonucleotide optionally targeting the first transcript or DNA locus; and

[0239] (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeted to the second transcript or DNA locus, wherein the second plurality differs from the first plurality in that at least one of the oligonucleotides present in the second plurality is labeled with a detectable moiety that is different from the corresponding oligonucleotide in the first plurality that targets the same transcript or DNA locus, such that in the second plurality:

[0240] (iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, or the third detectable moiety; and

[0241] (iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety, or the fourth detectable moiety, wherein the third oligonucleotide is labeled with a different detectable moiety than that used for the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than that used for the second oligonucleotide, or both;

[0242] (d) imaging the cell after said second contacting step to detect hybridization of said second plurality of oligonucleotides to their targets; and

[0243] (e) optionally repeating the contacting and imaging steps, each time using a new plurality of detectably labeled oligonucleotides, the new plurality of detectably labeled oligonucleotides comprising oligonucleotides that target overlapping transcripts or DNA loci targeted by the first plurality and the second plurality, wherein each plurality utilized differs from every other plurality utilized due to at least one difference in the detectable portion of the nucleotides that label the same transcript or DNA locus.

[0244] As used herein, detectably labeled oligonucleotides are labeled with a detectable moiety. In some embodiments, detectably labeled oligonucleotides comprise a detectable moiety. In some embodiments, detectably labeled oligonucleotides comprise two or more detectable moieties. In some embodiments, detectably labeled oligonucleotides have a detectable moiety. In some embodiments, detectably labeled oligonucleotides have two or more detectable moieties.

[0245] In some embodiments, the detectable portion is a fluorophore or comprises a fluorophore. Exemplary detectably labeled oligonucleotides labeled with fluorophores include, but are not limited to, probes for fluorescence in situ hybridization (FISH). As widely known and practiced by those of ordinary skill in the art, FISH is used to, among other things, detect and locate the presence or absence of specific DNA sequences or RNA targets. Methods for designing and preparing labeled detectably labeled oligonucleotides are widely known in the art, including, but not limited to, those methods described in U.S. Patent Application Publication US20120142014. However, due to limitations such as the availability of fluorophores, FISH can only be used for the analysis of a limited number of target profiles in a given experiment. By barcoding a variety of different target sequences, the methods provided by the present invention can be used for a large number of targets, up to F N , spectral analysis, where F is the number of types of detectable moieties (fluorophores in the case of FISH) and N is the number of contact steps (hybridization in the case of FISH). For example, when F is 4 and N is 8, almost the entire transcriptome (4 8 =65,536) can be spectrally analyzed. In some embodiments, F is at least 2. In some embodiments, F is 3. In some embodiments, F is 4. In some embodiments, F is 5. In some embodiments, F is 6. In some embodiments, F is 7. In some embodiments, F is 8. In some embodiments, F is 9. In some embodiments, F is 10. In some embodiments, F is 11. In some embodiments, F is 12. In some embodiments, F is 13. In some embodiments, F is 14. In some embodiments, F is 15. In some embodiments, F is greater than 15. In some embodiments, N is 2. In some embodiments, N is greater than 2. In some embodiments, N is 3. In some embodiments, N is greater than 3. In some embodiments, N is 4. In some embodiments, N is greater than 4. In some embodiments, N is 5. In some embodiments, N is greater than 5. In some embodiments, N is 6. In some embodiments, N is greater than 6. In some embodiments, N is 7. In some embodiments, N is greater than 7. In some embodiments, N is 8. In some embodiments, N is greater than 8. In some embodiments, N is 9. In some embodiments, N is greater than 9. In some embodiments, N is 10. In some embodiments, N is greater than 10. In some embodiments, the plurality of detectably labeled oligonucleotides targets at least 100 targets.

[0246] In the contacting step, the detectably labeled oligonucleotide can be labeled before, at the same time, or after it is combined with its target. In some embodiments, the detectably labeled oligonucleotide, such as the oligonucleotide of the labeled fluorophore, is labeled before it is combined with its target. In some embodiments, the detectably labeled oligonucleotide is labeled while it is combined with its target. In some embodiments, the detectably labeled oligonucleotide is labeled after it is combined with its target. In some embodiments, the detectably labeled oligonucleotide is labeled after it is combined with its target. In some embodiments, the detectably labeled oligonucleotide is labeled by orthogonal amplification with hybridization chain reaction (HCR) after hybridization (Choi, HM., Nat Biotechnol. 201011 month; 28 (ll): 1208-12). In some embodiments, the detectably labeled oligonucleotide comprises a part, such as a nucleic acid sequence, and the one or more parts that can provide a signal in the imaging step can be directly or indirectly connected to the oligonucleotide.

[0247] In some embodiments, the same type of label can be attached to different probes for different targets. In some embodiments, among the multiple detectably labeled oligonucleotides used in the contacting step, the probes for the same target have the same label (a set of detectably labeled oligonucleotides). After several rounds of contacting and imaging, each target has its own unique label combination (sequential barcode), so information about the target can be obtained, such as quantitative information and / or spatial information. For example, when a fluorophore is used to label a detectably labeled oligonucleotide, after N steps, the target will have a sequential barcode F1F2...F N , where F n is the color of the fluorophore used for the target in the nth imaging. One target can be distinguished from another due to the difference in their barcodes (e.g., red-red-blue-red compared to red-red-red-blue).

[0248] In some embodiments, the label of the present invention is or comprises one or more fluorescent dyes, including but not limited to fluorescein, rhodamine, Alexa Fluor type, Dylight Fluor type, ATTO type dyes, or any analogs or derivatives thereof.

[0249] In some embodiments, the labels of the present invention include but are not limited to fluorescein and chemical derivatives of fluorescein; eosin; carboxyfluorescein; fluorescein isothiocyanate (FITC); fluorescein amidite (FAM); erythrosine; rose bengal; fluorescein secreted from the bacterium Pseudomonas aeruginosa; methylene blue; laser dyes, rhodamine dyes (e.g., rhodamine, rhodamine 6G, rhodamine B, rhodamine 123, auramine O, sulforhodamine 101, sulforhodamine B and Texas Red).

[0250] In some embodiments, the labels of the present invention include but are not limited to ATTO dyes; acridine dyes (e.g., acridine orange, acridine yellow); Alexa Fluor; 7-aminoactinomycin D; 8-anilinonaphthalene-l-sulfonate; auramine-rhodamine dyes; benzanthrone; 5,12-bis(phenylethynyl)tetracene; 9,10-bis(phenylethynyl)anthracene; Blacklight paint; Brainbow; calcein; carboxyfluorescein; carboxydiacetate fluorescein succinimidyl ester; carboxyfluorescein succinimidyl ester; l-chloro-9,10-bis(phenylethynyl)anthracene; 2-chloro-9,10-bis(phenylethynyl)anthracene; 2-chloro-9,10-diphenylanthracene; coumarins; cyanine dyes (e.g., cyanines such as Cy3 and Cy5, DiOC6, SYBR Green I); DAPI, Darkquencher, Dylight Fluor, Fluo-4, FluoProbes; fluorochromes (e.g., calcein, carboxyfluorescein, carboxyfluorescein diacetate succinimidyl ester, carboxyfluorescein succinimidyl ester, eosin, eosin B, eosin Y, erythrosine, fluorescein, fluorescein isothiocyanate, fluorescein amide, Indian yellow, mercurochrome); Fluoro-Jade dyes; Fura-2; Fura-2-acetoxymethyl ester; green fluorescent protein, Hoec hst dyes, Indian yellow, Indo-1, fluorescent yellow, fluorescein, merocyanine, optical brighteners, oxazine dyes (e.g., cresyl violet, Nile blue, Nile red); perylene; phenanthridinium dyes (ethidium bromide and propidium iodide); phloxanthine dyes, phycobilins, phycoerythrin, phycoerythrin, 8-hydroxy-1,3,6-trisulfonic acid pyrene (Pyranine), rhodamine, rhodamine 123, rhodamine 6G, RiboGreen, RoGFP, rubrene, SYBR Green I, (E)-stilbene, (Z)-stilbene, sulforhodamine 101, sulforhodamine B, Synapto-pHluorin, tetraphenylbutadiene, Tetrasodium tris(bathophenanthroline disulfonate)ruthenium(II), Texas Red, TSQ, umbelliferone, or yellow fluorescent protein.

[0251] In some embodiments, the labels of the present invention include but are not limited to the AlexaFluor family of fluorescent dyes (Molecular Probes, Oregon). In fluorescence microscopy and cell biology, AlexaFluor dyes are widely used as cell and tissue markers. The excitation and emission spectra of the Alexa Fluor series cover the visible spectrum and extend to the infrared spectrum. They are numbered roughly according to the maximum excitation (in nm) of the individual members of the family. Some Alexa Fluor dyes are synthesized by sulfonation of coumarins, rhodamines, xanthenes (such as fluorescein) and cyanine dyes. In some embodiments, sulfonation makes Alexa Fluor dyes negatively charged and hydrophilic. In some embodiments, Alexa Fluor dyes are more stable, brighter and less sensitive to pH than ordinary dyes (e.g., fluorescein, rhodamine) with comparable excitation and emission, and are to some extent newer cyanine series. Exemplary Alexa Fluor dyes include, but are not limited to, Alexa-350, Alexa-405, Alexa-430, Alexa-488, Alexa-500, Alexa-514, Alexa-532, Alexa-546, Alexa-555, Alexa-568, Alexa-594, Alexa-610, Alexa-633, Alexa-647, Alexa-660, Alexa-680, Alexa-700, or Alexa-750.

[0252] In some embodiments, the label of the invention comprises one or more of the DyLight Fluor family of fluorescent dyes (Dyomics and Thermo Fisher Scientific). Exemplary AlexaFluor family dyes include, but are not limited to, DyLight-350, DyLight-405, DyLight-488, DyLight-549, DyLight-594, DyLight-633, DyLight-649, DyLight-680, DyLight-750, or DyLight-800.

[0253] In some embodiments, the detectable moiety is or comprises a nanomaterial. In some embodiments, the detectable moiety is or comprises a nanoparticle. In some embodiments, the detectable moiety is or comprises a quantum dot. In some embodiments, the detectable moiety is a quantum dot. In some embodiments, the detectable moiety comprises a quantum dot. In some embodiments, the detectable moiety is or comprises a gold nanoparticle. In some embodiments, the detectable moiety is a gold nanoparticle. In some embodiments, the detectable moiety comprises a gold nanoparticle.

[0254] In some embodiments, one of skill in the art understands that the choice of label for a particular probe in a particular cycle can be determined based on a variety of factors including, for example, size, type of signal generated, manner of attachment to or incorporation into the probe, properties of the cellular component including location of the cellular component within the cell, properties of the cell, type of interaction being analyzed, and the like.

[0255] For example, in some embodiments, the probe is labeled with Cy3 or Cy5, which is synthesized to carry an N-hydroxysuccinimide ester (NHS-ester) reactive group. Since NHS-esters react easily with aliphatic amine groups, nucleotides can be modified with aminoalkyl groups. This can be done by incorporating aminoalkyl-modified nucleotides during the synthesis reaction. In some embodiments, one label is used for every 60 bases to avoid quenching.

[0256] Detectably labeled oligonucleotides can hybridize with a target, such as a transcript or a DNA locus. In some embodiments, the target is or comprises a transcript. In some embodiments, the target is a transcript. In some embodiments, the transcript is RNA. In some embodiments, the transcript is mRNA. In some embodiments, the transcript is tRNA. In some embodiments, the transcript is rRNA. In some embodiments, the transcript is snRNA. In some embodiments, RNA is non-coding RNA. Exemplary non-coding RNA types are widely known in the art, including but not limited to long non-coding RNA (lncRNA), microRNA (miRNA), short interfering RNA (siRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNA) and other short RNAs. In some embodiments, RNA is IncRNA. In some embodiments, RNA is miRNA. In some embodiments, RNA is piRNA. In some embodiments, RNA is snoRNA.

[0257] In some embodiments, the target is or comprises a DNA locus. In some embodiments, when the target is a DNA locus, the detectably labeled oligonucleotide optionally comprises one or more RNA nucleotides or RNA segments. The detectably labeled oligonucleotide comprises an RNA sequence that can be selectively removed after imaging without degrading the DNA target, for example, by digestion with an RNA-specific enzyme. Exemplary enzymes that specifically degrade RNA without degrading DNA include, but are not limited to, a variety of RNases, such as RNase A and RNase H.

[0258] In some embodiments, detectably labeled oligonucleotides are directly hybridized with their targets, such as transcripts or DNA loci. In some embodiments, detectably labeled oligonucleotides interact specifically with their targets (specifically identifying their targets) by combining or hybridizing with one or more intermediates, and the one or more intermediates (such as oligonucleotides) are combined with, hybridized or otherwise specifically connected to the target. In some embodiments, the intermediate oligonucleotides are hybridized with their targets with overhangs, so the second oligonucleotides ("bridge oligonucleotides" or "bridge probes") with complementary sequences can be combined with the intermediate oligonucleotides. In some embodiments, the intermediates target nucleic acids and are optionally labeled with detectable moieties, and after hybridizing with the target, overhang sequences are included. In some embodiments, the intermediates include sequences, overhang sequences, and optionally detectable moieties hybridized with the target. In some embodiments, the intermediates include sequences and overhang sequences hybridized with the target. In some embodiments, the intermediates do not have detectable moieties. In some embodiments, the second oligonucleotides are detectably labeled oligonucleotides. In some embodiments, the second detectably labeled oligonucleotides are labeled with dyes. In some embodiments, the detectably labeled oligonucleotide is labeled by HCR polymer. In some embodiments, throughout multiple contacts, removal and / or imaging steps, the intermediate oligonucleotide remains combined with the target; In contact and imaging steps, a sequential barcode is provided by a combination of detectable labels, and the detectable label is connected to the intermediate oligonucleotide by a bridging probe. For example, when the detectably labeled oligonucleotide is used as a bridging probe, a barcode is provided by the detectably labeled oligonucleotide, and the detectably labeled oligonucleotide is hybridized with the intermediate oligonucleotide by their overhang sequence. After the imaging step, the bridging oligonucleotide is optionally removed as described herein. In some embodiments, 1 intermediate oligonucleotide is used for a target. In some embodiments, 2 or more intermediate oligonucleotides are used for a target. In some embodiments, 3 or more intermediate oligonucleotides are used for a target. In some embodiments, 4 or more intermediate oligonucleotides are used for a target. In some embodiments, 5 or more intermediate oligonucleotides are used for a target. In some embodiments, 6 or more intermediate oligonucleotides are used for a target. In some embodiments, 7 or more intermediate oligonucleotides are used for one target. In some embodiments, 8 or more intermediate oligonucleotides are used for one target. In some embodiments, 9 or more intermediate oligonucleotides are used for one target. In some embodiments, 10 or more intermediate oligonucleotides are used for one target. In some embodiments, 11 or more intermediate oligonucleotides are used for one target. In some embodiments, 12 or more intermediate oligonucleotides are used for one target. In some embodiments, 13 or more intermediate oligonucleotides are used for one target.In some embodiments, 14 or more intermediate oligonucleotides are used for one target. In some embodiments, 15 or more intermediate oligonucleotides are used for one target. In some embodiments, 16 or more intermediate oligonucleotides are used for one target. In some embodiments, 17 or more intermediate oligonucleotides are used for one target. In some embodiments, 18 or more intermediate oligonucleotides are used for one target. In some embodiments, 19 or more intermediate oligonucleotides are used for one target. In some embodiments, 20 or more intermediate oligonucleotides are used for one target. In some embodiments, 21 or more intermediate oligonucleotides are used for one target. In some embodiments, 22 or more intermediate oligonucleotides are used for one target. In some embodiments, 23 or more intermediate oligonucleotides are used for one target. In some embodiments, 24 or more intermediate oligonucleotides are used for one target. In some embodiments, 25 or more intermediate oligonucleotides are used for one target. In some embodiments, 30 or more intermediate oligonucleotides are used for one target. In some embodiments, 40 or more intermediate oligonucleotides are used for one target. In some embodiments, 50 or more intermediate oligonucleotides are employed for one target.

[0259] In some embodiments, each intermediate oligonucleotide hybridizes with a different sequence of the target. In some embodiments, each intermediate oligonucleotide of the target comprises the same overhang sequence. In some embodiments, each detectably labeled oligonucleotide for the target comprises the same sequence complementary to the same overhang sequence common to all intermediate oligonucleotides of the target. In some embodiments, the intermediate oligonucleotide comprises a sequence complementary to the target and a sequence complementary to the detectably labeled oligonucleotide.

[0260] In some embodiments, the provided methods further include the following steps:

[0261] (f) performing a contacting step comprising contacting a cell comprising a plurality of nucleic acids with a plurality of intermediate oligonucleotides, each of the plurality of intermediate oligonucleotides:

[0262] (i) targets a nucleic acid and is optionally labeled with a detectable moiety; and

[0263] (ii) comprises an overhang sequence after hybridization to the target; and

[0264] (g) optionally imaging the cells to detect the interaction between the intermediary oligonucleotide and its target.

[0265] In some embodiments, step (f) and optional step (g) are performed before step (a). In some embodiments, step (f) is step (a) that is performed. In some embodiments, the removal step retains the intermediate oligonucleotide.

[0266] In some embodiments, the technology provided is used for different transcript profiles formed due to splicing variation, RNA editing, oligonucleotide modification or a combination thereof. In some embodiments, the target is an RNA splice variant. In some embodiments, the technology provided is for one or more splice variants of a gene, such as one or more splice variants of a gene, and the position and quantity spectrum analysis. In some embodiments, the method or composition provided is for different splice variant spectrum analysis. In some embodiments, the exons comprising one or more variants are targeted and barcoded by sequential hybridization and barcoding. In some embodiments, the splice variants include one or more distinguishable sequences obtained by splicing, and such sequences are targeted. In some embodiments, by targeting exons and / or distinguishable sequences, the technology provided can be for one or more specific splice variants or mRNA The entire splicing library (splicing repertoire) spectrum analysis. As widely known in the art, mRNA splicing is important for many biological processes and diseases, for example, neurological diseases, such as autism or Down syndrome. Molecules responsible for cell-cell adhesion and synaptogenesis are spliced ​​and their defects in the brain are known to produce miswiring and lead to disease.

[0267] In some embodiments, detectably labeled oligonucleotides target sequence modifications caused by sequence editing, chemical modification and / or a combination thereof. In some embodiments, optionally after the conversion process, the modified nucleic acid target is hybridized with one or more different complementary sequences compared to the unmodified target, and one or more oligonucleotides selectively hybridized with the modified nucleic acid are analyzed by spectrum. In some embodiments, the target is RNA edited by RNA (Brennicke, A., A.Marchfelder, et al. (1999). "RNA editing". FEMS Microbiol Rev 23 (3): 297-316). In some embodiments, the provided technology analyzes the different RNA variants formed by RNA editing. In some embodiments, the provided technology analyzes the oligonucleotide spectrum of modification. In some embodiments, the provided technology analyzes the RNA spectrum of methylation (Song CX, Yi C, He C. Mapping recently identified nucleotide variants in the genome and transcriptome. Nat Biotechnol. 2012 November; 30 (l 1): 1107-16). In certain embodiments, provided techniques profile methylated DNA. In some embodiments, the target is a single nucleotide polymorphism (SNP).

[0268] In some embodiments, by profiling the targets, the provided techniques provide, among other things, information on the number and / or location of targets in some cases in a single cell, tissue, organ, or organism. In some embodiments, profiling of transcripts can be used to qualitatively and / or quantitatively define the spatiotemporal patterns of gene expression in a cell, tissue, organ, or organism.

[0269] In some embodiments, each detectably labeled oligonucleotide in a group has different targets, for example, transcripts or DNA loci. In some embodiments, two or more detectably labeled oligonucleotides in a group have the same target. In some embodiments, two or more detectably labeled oligonucleotides target the same transcript. In some embodiments, two or more detectably labeled oligonucleotides target the same DNA locus. In some embodiments, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 detectably labeled oligonucleotides target the same target. In some embodiments, two or more detectably labeled oligonucleotides target the same target. In some embodiments, 5 or more detectably labeled oligonucleotides target the same target. In some embodiments, 10 or more detectably labeled oligonucleotides target the same target. In some embodiments, 15 or more detectably labeled oligonucleotides target the same target. In some embodiments, 20 or more detectably labeled oligonucleotides target the same target. In some embodiments, 25 or more detectably labeled oligonucleotides target the same target. In some embodiments, 30 or more detectably labeled oligonucleotides target the same target. In some embodiments, 35 or more detectably labeled oligonucleotides target the same target. In some embodiments, 40 or more detectably labeled oligonucleotides target the same target. In some embodiments, 45 or more detectably labeled oligonucleotides target the same target. In some embodiments, 50 or more detectably labeled oligonucleotides target the same target. In some embodiments, 60 or more detectably labeled oligonucleotides target the same target. In some embodiments, 70 or more detectably labeled oligonucleotides target the same target. In some embodiments, 80 or more detectably labeled oligonucleotides target the same target. In some embodiments, 90 or more detectably labeled oligonucleotides target the same target. In some embodiments, 100 or more detectably labeled oligonucleotides target the same target. In some embodiments, about 1-10 detectably labeled oligonucleotides target the same target. In some embodiments, about 5-15 detectably labeled oligonucleotides target the same target. In some embodiments, about 10-20 detectably labeled oligonucleotides target the same target. In some embodiments, about 15-25 detectably labeled oligonucleotides target the same target. In some embodiments, about 20-30 detectably labeled oligonucleotides target the same target.In some embodiments, about 25-35 detectably labeled oligonucleotides target the same target. In some embodiments, about 30-40 detectably labeled oligonucleotides target the same target. In some embodiments, about 35-45 detectably labeled oligonucleotides target the same target. In some embodiments, about 40-50 detectably labeled oligonucleotides target the same target. In some embodiments, about 45-55 detectably labeled oligonucleotides target the same target. In some embodiments, about 50-70 detectably labeled oligonucleotides target the same target. In some embodiments, about 60-80 detectably labeled oligonucleotides target the same target. In some embodiments, about 70-90 detectably labeled oligonucleotides target the same target. In some embodiments, about 80-100 detectably labeled oligonucleotides target the same target.

[0270] In some embodiments, use of multiple detectably labeled oligonucleotides to the same target increases signal strength, among other things. In some embodiments, each detectably labeled oligonucleotide in a group targeting the same target interacts with a different portion of the target.

[0271] In some embodiments, all detectably labeled oligonucleotides for a target in a group have the same detectable moiety. In some embodiments, all detectably labeled oligonucleotides are labeled in the same manner. In some embodiments, all detectably labeled oligonucleotides for a target have the same fluorophore.

[0272] In some embodiments, the detectably labeled oligonucleotide for the target is positioned in the targeted region of the target. The targeted region can have a variety of lengths. In some embodiments, the length of the targeted region is about 20bp. In some embodiments, the length of the targeted region is about 30bp. In some embodiments, the length of the targeted region is about 40bp. In some embodiments, the length of the targeted region is about 50bp. In some embodiments, the length of the targeted region is about 60bp. In some embodiments, the length of the targeted region is about 80bp. In some embodiments, the length of the targeted region is about 100bp. In some embodiments, the length of the targeted region is about 150bp. In some embodiments, the length of the targeted region is about 200bp. In some embodiments, the length of the targeted region is about 250bp. In some embodiments, the length of the targeted region is about 300bp. In some embodiments, the length of the targeted region is about 350bp. In some embodiments, the length of the targeted region is about 400bp. In some embodiments, the length of the targeted region is about 450bp. In some embodiments, the length of the targeted region is about 500bp. In some embodiments, the length of the targeted region is about 600bp. In some embodiments, the length of the targeted region is about 700bp. In some embodiments, the length of the targeted region is about 800bp. In some embodiments, the length of the targeted region is about 900bp. In some embodiments, the length of the targeted region is about 1,000bp. In some embodiments, the detectably labeled oligonucleotides for the target are positioned adjacent to each other on the target.

[0273] As will be appreciated by those of ordinary skill in the art, different techniques may be used for the imaging step. Exemplary methods include, but are not limited to, epifluorescence microscopy, confocal microscopy, different types of super-resolution microscopy (PALM / STORM, SSIM / GSD / STED), and light sheet illumination microscopy (SPIM, etc.).

[0274] Exemplary super-resolution techniques include, but are not limited to: 5M and 4Pi-microscopy, stimulated emission depletion microscopy (STEDM), ground state depletion microscopy (GSDM), spatial structured illumination microscopy (SSIM), photoactivated localization microscopy (PALM), reversible saturated light linear fluorescence transition (RESOLFT), total internal reflection fluorescence microscopy (TIRFM), fluorescence-PALM (FPALM), stochastic optical reconstruction microscopy (STORM), fluorescence imaging with one nanometer accuracy (FIONA) and their combinations. For example: Chi, 2009, "Super-resolution microscopy: breaking the limits, Nature Methods6(1): 15-18; Blow2008, "New ways to see a smaller world," Nature 456: 825-828; Hell, et al., 2007, "Far-Field Optical Nanoscopy,"Science316:1153; R.Heintzmann and G.Ficz, 2006,"Breaking the resolution limit in light microscopy,"Briefings in Functional Genomicsand Proteomics 5(4):289-301; Garini et al., 2005,"From micro to nano:recent advances in high-resolution microscopy,"Current Opinion in Biotechnology 16:3-12; and Bewersdorf et al., 2006, "Comparison of I 5 M and 4Pi-microscopy," 222(2):105-117; and Wells, 2004, "Man the Nanoscopes," JCB 164(3):337-340.

[0275] In some embodiments, electron microscopy (EM) is used.

[0276] In some embodiments, the imaging step detects the target. In some embodiments, the imaging step locates the target. In some embodiments, the imaging step provides three-dimensional spatial information of the target. In some embodiments, the imaging step quantifies the target. By using multiple contacting and imaging steps, the provided methods can provide spatial information and / or quantitative information for a large number of targets with unexpectedly high throughput. For example, when F different detectable labels are used, up to F scales can be obtained after N contacting and imaging steps. N The spatial information and / or quantitative information of each target.

[0277] In some embodiments, the provided method includes additional steps before or after the contact and / or imaging steps. In some embodiments, the provided method includes the step of removing a plurality of detectably labeled oligonucleotides after each imaging step. In some embodiments, the removal step includes degrading the detectably labeled oligonucleotides. In some embodiments, the removal step does not significantly degrade the target, so that if desired, the target can be used for the next round of contact and / or imaging steps. In some embodiments, the removal step includes contacting a plurality of detectably labeled oligonucleotides with an enzyme that digests the detectably labeled oligonucleotides. In some embodiments, the removal step includes contacting the detectably labeled oligonucleotides with a DNA enzyme or an RNA enzyme. For example, in some embodiments, the detectably labeled oligonucleotides include a DNA sequence and a DNA enzyme is used for its degradation; in some other embodiments, the detectably labeled oligonucleotides include an RNA sequence and an RNA enzyme is used for its degradation. In some embodiments, the removal step includes degrading the detectable portion. In some embodiments, the removal step includes photobleaching.

[0278] In some embodiments, the target of one group of detectably labeled oligonucleotides is also the target of another group. In some embodiments, the target of one group of detectably labeled oligonucleotides overlaps with the target of another group. In some embodiments, the overlap is more than 10%. In some embodiments, the overlap is more than 20%. In some embodiments, the overlap is more than 30%. In some embodiments, the overlap is more than 40%. In some embodiments, the overlap is more than 50%. In some embodiments, the overlap is more than 60%. In some embodiments, the overlap is more than 70%. In some embodiments, the overlap is more than 80%. In some embodiments, the overlap is more than 90%. In some embodiments, the overlap is more than 91%. In some embodiments, the overlap is more than 92%. In some embodiments, the overlap is more than 93%. In some embodiments, the overlap is more than 94%. In some embodiments, the overlap is more than 90%. In some embodiments, the overlap is more than 95%. In some embodiments, the overlap is more than 96%. In some embodiments, the overlap is more than 97%. In some embodiments, the overlap is more than 98%. In some embodiments, the overlap is more than 99%. In some embodiments, the overlap is more than 99.5%. In some embodiments, the overlap is more than 99.6%. In some embodiments, the overlap is more than 99.7%. In some embodiments, the overlap is more than 99.8%. In some embodiments, the overlap is more than 99.9%. In some embodiments, the overlap is 100%. In some embodiments, the target of one group of detectably labeled oligonucleotides is the same as the target of another group. In some embodiments, each group of detectably labeled oligonucleotides targets the same target.

[0279] In some embodiments, the third detectably labeled oligonucleotide targeting the first transcript or DNA locus (first target) in the second contacting step optionally has the same sequence as the first detectably labeled oligonucleotide targeting the first transcript or DNA locus. In some embodiments, the sequences are identical. In some embodiments, the sequences are different. Similarly, in some embodiments, the fourth detectably labeled oligonucleotide targeting the second transcript or DNA locus (first target) in the second contacting step optionally has the same sequence as the second detectably labeled oligonucleotide targeting the first transcript or DNA locus. In some embodiments, the sequences are identical. In some embodiments, the sequences are different.

[0280] In some embodiments, the second plurality differs from the first plurality in that at least one of the oligonucleotides present in the second plurality is labeled with a detectable moiety that is different from a corresponding oligonucleotide in the first plurality that targets the same transcript or DNA locus. In some embodiments, each plurality of a plurality of detectably labeled oligonucleotides differs from another plurality in that at least one oligonucleotide present in one plurality is labeled with a detectable moiety that is different from a corresponding oligonucleotide in the other plurality that targets the same transcript or DNA locus.

[0281] In some embodiments, the detectably labeled oligonucleotide has a [S]-[L] structure, wherein [S] is an oligonucleotide sequence and [L] is a detectable moiety or a combination of detectable moieties. In some embodiments, [L] comprises a plurality of units of a detectable label, such as a fluorophore, each of which independently associates with one or more nucleoside moieties of an oligonucleotide sequence, such as [S]. In some embodiments, each detectable label attached to the same detectably labeled oligonucleotide provides the same detectable signal. In some embodiments, all detectable labels attached to the same oligonucleotide sequence are the same.

[0282] In some embodiments, the oligonucleotides targeting the same target in two or more sets of detectably labeled oligonucleotides have the same set of sequences, i.e., the differences between the detectably labeled oligonucleotides, if any, are in the detectable moiety, not in the sequence. For example, in a set of detectably labeled oligonucleotides, the detectably labeled oligonucleotides targeting a first target all have the same detectable moiety, or combination of detectable moieties [L]1:

[0283] [S]1-[L]1, [S]2-[L]1,…,[S] n -[L] 1, wherein n is the number of detectably labeled oligonucleotides directed against a target, e.g., an integer from 3 to 50;

[0284] In another set of detectably labeled oligonucleotides, wherein oligonucleotides directed to the same target are differently labeled, the oligonucleotides directed to the same target have the same set of oligonucleotide sequences ([S]1, [S]2, ..., [S] n ), and a different [L]2:

[0285] [S]1-[L]2, [S]2-[L]2,…,[S] n -[L]2, where [L]1 is detected differently from [L]2.

[0286] To illustrate certain embodiments of the present invention, the following two-step, two-label, four-target (F N =2 2=4) process, wherein all detectably labeled oligonucleotides targeting the same target in each group independently have the same detectable moiety:

[0287] Step 1. Contacting the target with a first plurality (P1) of detectably labeled oligonucleotides:

[0288] Target T1: [S] P1-T1-1 [L]1, [S] P1-T1-2 [L]1, [S] P1-T1-3 [L] 1, ..., [S] P1-T1-P1T1 [L]1, wherein P1T1 is the number of detectably labeled oligonucleotides in the first plurality that target T1, and [L]1 is a first detectable label;

[0289] Target T2: [S] P1-T2-1 [L]1, [S] P1-T2-2 [L]1, [S] P1-T2-3 [L] 1, ..., [S] P1-T2-P1T2 [L] 1, wherein P1T2 is the number of detectably labeled oligonucleotides targeting T2 in the first plurality;

[0290] Target T3: [S] P1-T3-1 [L]2, [S] P1-T3-2 [L]2, [S] P1-T3-3 [L]2, ..., [S] P1-T3-PIT3 [L]2, wherein P1T3 is the number of detectably labeled oligonucleotides targeting T3 in the first plurality, and [L]2 is a different detectable label than [L]1;

[0291] Target T4: [S] P1-T4-1 [L]2, [S] P1-T4-2 [L]2, [S] P1-T4-3 [L]2, ..., [S] P1-T4-PIT4 [L] 2, wherein P1T4 is the number of detectably labeled oligonucleotides targeting T4 in the first plurality.

[0292] Step 2: Imaging;

[0293] Step 3: Removing P1 from the target;

[0294] Step 4: Contacting the target with a second plurality (P2) of detectably labeled oligonucleotides:

[0295] Target T1: [S] P2-T1-1 [L]1, [S] P2-T1-2 [L]1, [S] P2-T1-3 [L] 1, ..., [S] P2-T1-P2T1[L] 1, wherein P2T1 is the number of detectably labeled oligonucleotides targeting T1 in the second plurality;

[0296] Target T2: [S] P2-T2-1 [L]2, [S] P2-T2-2 [L]2, [S] P2-T2-3 [L]2, ..., [S] P2-T2-P2T2 [L] 2, wherein P2T2 is the number of detectably labeled oligonucleotides targeting T2 in the second plurality;

[0297] Target T3: [S] P2-T3-1 [L]1, [S] P2-T3-2 [L]1, [S] P2-T3-3 [L] 1, ..., [S] P2-T3-P2T3 [L] 1, wherein P2T3 is the number of detectably labeled oligonucleotides targeting T3 in the second plurality;

[0298] Target T4: [S] P2-T4-1 [L]2, [S] P2-T4-2 [L]2, [S] P2-T4-3 [L]2, ..., [S] P2-T4-P2T4 [L] 2, wherein P2T4 is the number of detectably labeled oligonucleotides targeting T4 in the second plurality.

[0299] Step 5: Imaging.

[0300] After two imaging steps, each target has its own unique sequential barcode:

[0301] T1: [L]1[L]1;

[0302] T2: [L]1[L]2;

[0303] T3: [L]2[L]1; and

[0304] T4: [L]2[L]2.

[0305] In some embodiments, additional barcodes, Tl--, T2--, --Tl, --T2 may also be used, where -- indicates no signal for that step.

[0306] In the above exemplary process, each of P1T1, P1T2, P1T3, P1T4, P2T1, P2T2, P2T3 and P2T4 is independently a natural number (an integer greater than 0). In some embodiments, P1T1=P2T1. In some embodiments, P1T2=P2T2. In some embodiments, P1T3=P2T3. In some embodiments, P1T4=P2T4. In some embodiments, one detectably labeled oligonucleotide is used for the target. In some embodiments, two or more detectably labeled oligonucleotides are used for the target.

[0307] In some embodiments, the detectably labeled oligonucleotides targeting the same target in each plurality have the same sequence set. For example, for target T1 in the above example, [S] P1-T1-1 To [S] P1_T1_P1T1 Each of which independently has the same P2-T1-1 To [S] P2-TI-P2TI one of the same sequence, and [S] P2-T1-1 To [S] P2-T1-P2T1 Each of which independently has the same P1-T1-1 To [S] P1-TI-P1T1 In some embodiments, the detectably labeled oligonucleotides directed to the same target in each plurality have different sets of sequences.

[0308] In some embodiments, the provided methods optionally include a step of removing the plurality of detectably labeled oligonucleotides after the imaging step. In some embodiments, the provided methods include a removal step after the imaging step. In some embodiments, the provided methods include a removal step after each imaging step except the last imaging step. In some embodiments, the provided methods include a removal step after each imaging step.

[0309] The removal step in the provided method can achieve one or more of a variety of purposes. In some embodiments, the removal step removes a variety of detectably labeled oligonucleotides from the target so that the target can be used to interact with another variety of detectably labeled oligonucleotides. In some embodiments, the removal step removes a variety of detectably labeled oligonucleotides so that the detectable portion of a variety of detectably labeled oligonucleotides does not interfere with the detection of the combination of another variety of detectably labeled oligonucleotides with the target. In some embodiments, the removal step removes at least 80% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 85% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 90% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 91% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 92% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 93% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 94% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 95% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 96% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 97% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 98% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 99% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 99.1% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 99.2% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 99.3% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 99.4% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 99.5% of the detectably labeled oligonucleotides. In some embodiments, the removal step removes at least 80% of the detectable signal. In some embodiments, the removal step removes at least 85% of the detectable signal. In some embodiments, the removing step removes at least 90% of the detectable signal. In some embodiments, the removing step removes at least 91% of the detectable signal. In some embodiments, the removing step removes at least 92% of the detectable signal. In some embodiments, the removing step removes at least 93% of the detectable signal. In some embodiments, the removing step removes at least 94% of the detectable signal. In some embodiments, the removing step removes at least 95% of the detectable signal. In some embodiments, the removing step removes at least 96% of the detectable signal.In some embodiments, the removing step removes at least 97% of the detectable signal. In some embodiments, the removing step removes at least 98% of the detectable signal. In some embodiments, the removing step removes at least 99% of the detectable signal. In some embodiments, the removing step removes at least 99.5% of the detectable signal. In some embodiments, the removing step removes 100% of the detectable signal. In some embodiments, no signal is detectable by the imaging step after the removing step.

[0310] The removal step optionally retains the target (e.g., transcript or DNA locus) for further use, e.g., further detection or quantification by additional contacting and / or imaging steps. In some embodiments, the removal step retains at least 80% of the target. The percentage of the target retained can be measured, for example, by comparing the data collected before and after the removal step, optionally using the same contact and imaging scheme. In some embodiments, the removal step retains at least 85% of the target. In some embodiments, the removal step retains at least 90% of the target. In some embodiments, the removal step retains at least 91% of the target. In some embodiments, the removal step retains at least 92% of the target. In some embodiments, the removal step retains at least 93% of the target. In some embodiments, the removal step retains at least 94% of the target. In some embodiments, the removal step retains at least 95% of the target. In some embodiments, the removal step retains at least 96% of the target. In some embodiments, the removal step retains at least 97% of the target. In some embodiments, the removal step retains at least 98% of the target. In some embodiments, the removal step retains at least 99% of the target.

[0311] The method for removing detectably labeled oligonucleotide is widely known in the field. In some embodiments, the removal step comprises degrading the detectably labeled oligonucleotide. In some embodiments, the detectably labeled oligonucleotide is removed by enzymatic digestion. In some embodiments, the removal step comprises contacting a plurality of detectably labeled oligonucleotides with an enzyme that digests the detectably labeled oligonucleotides.

[0312] Suitable enzymes are widely used in the art. For example, depending on the type of detectably labeled oligonucleotide and / or target, DNA enzyme or RNA enzyme can be used. In some embodiments, detectably labeled oligonucleotides comprising a DNA sequence for detecting / quantitative RNA target are digested by DNA enzymes such as DNA enzyme I. In some embodiments, detectably labeled oligonucleotides comprising an RNA sequence for detecting / quantitative DNA target are digested by RNA enzymes. In some embodiments, detectably labeled RNA oligonucleotides are used to target DNA loci.

[0313] In some embodiments, the detectably labeled oligonucleotide interacts with its target by combining or hybridizing with one or more intermediates, and the one or more intermediates are such as oligonucleotides combined with the target, hybridized or otherwise connected. In some embodiments, the detectably labeled oligonucleotide interacts with the target by hybridizing with an intermediate oligonucleotide, and the intermediate oligonucleotide hybridizes with the target, wherein the intermediate oligonucleotide comprises a sequence complementary to the target and a sequence (overhang) complementary to the detectably labeled oligonucleotide. In some embodiments, the removal step removes the detectably labeled oligonucleotide, and optionally the intermediate oligonucleotide is kept intact. In some embodiments, the removal step removes the detectably labeled oligonucleotide and the intermediate oligonucleotide is kept intact. In some embodiments, from a chemical or enzymology perspective, the detectably labeled oligonucleotide is different from the intermediate, so that the detectably labeled oligonucleotide can be selectively removed.

[0314] In some embodiments, the intermediate DNA oligonucleotide is used to hybridize with the DNA locus with an overhang (e.g., 20nt) so that the bridging oligonucleotide comprising the RNA sequence can be combined with a complementary sequence (e.g., RNA bridging probe). The RNA bridging probe can be directly labeled with a dye or HCR polymer (can also be DNA). After imaging, RNA enzymes can be used to digest the RNA bridging probe while keeping the DNA probe intact and hybridized on the DNA locus. Such a method provides multiple advantages. For example, the subsequent contact step only includes the RNA bridging probe hybridizing with the DNA oligonucleotide with an overhang, and avoids denaturing the double-stranded DNA and hybridizing with the DNA oligonucleotide, which is a difficult process. In addition, the overhangs of all DNA oligonucleotides (e.g., 20 to 40) targeting the same gene can be made the same, so that each round of hybridization only requires one type of RNA bridging probe per gene. In order to switch colors for different hybridizations (contact steps), technicians can change the RNA bridging probe with different labels or different HCR polymers. Such a DNA bridging probe can also be used: it can be specifically removed, for example, with a specific enzyme restriction site such as EcoRI on a bridge or HCR hairpin. Incubation of the cells with an appropriate nuclease can digest away all detectable moieties without affecting the DNA loci and / or probes hybridized thereto.

[0315] In some embodiments, the detectably labeled oligonucleotide comprises a 5'-phosphorylated and can be degraded by Lambda exonuclease, while the intermediate oligonucleotide is not 5'-phosphorylated and cannot be degraded by Lambda exonuclease.

[0316] In some embodiments, the detectably labeled oligonucleotide comprises uracil. In some embodiments, the detectably labeled oligonucleotide comprises uracil and can be USER TM The intermediate oligonucleotide does not contain uracil and cannot be degraded by USER TM Enzymatic degradation.

[0317] In some embodiments, the oligonucleotide hybridized with the overhang of the intermediate oligonucleotide has a recessed 3'-end when hybridizing with the overhang. The detectably labeled oligonucleotide with a recessed 3'-end can be selectively digested by exonuclease III when hybridizing with the intermediate oligonucleotide. Since exonuclease III is much less active to the intermediate oligonucleotide that does not have a recessed 3'-end or whose 3'-end is an RNA-DNA duplex, they can remain intact.

[0318] In some embodiments, when enzymes are involved, the removal step is performed at a temperature that produces the best results. In some embodiments, the removal step is performed at about 37°C. In some embodiments, the removal step is performed at room temperature. In some embodiments, digestion with Lambda exonuclease is performed at about 37°C. In some embodiments, the removal step is performed at room temperature. TM Enzymatic digestion is performed at about 37°C. In some embodiments, USER TM Enzymatic digestion is performed at room temperature. In some embodiments, digestion with Exonuclease III is performed at about 37° C. In some embodiments, digestion with Exonuclease III is performed at room temperature.

[0319] In some embodiments, the use of the overhang sequence of the oligonucleotide combination for detectably labeled intermediate oligonucleotide provides multiple advantages.In some embodiments, the course of hybridization between the overhang sequence and the oligonucleotide that can be detected marked is faster than the course of hybridization between the intermediate oligonucleotide and the target.In some embodiments, all intermediate oligonucleotides for target comprise the same overhang sequence, and all detectably labeled oligonucleotides for target comprise the same complementary sequence for being combined with the same overhang sequence.In some embodiments, the hybridization between the oligonucleotide that can be detected marked and one group of intermediate oligonucleotide is faster than the hybridization between one group of intermediate oligonucleotide and one group of target up to about 20-40 times.In some embodiments, the hybridization between the oligonucleotide that can be detected marked and the intermediate oligonucleotide can be completed in 30 minutes, compared with, in some cases, for the hybridization between the intermediate oligonucleotide and the target up to about 12 hours.

[0320] In some embodiments, strand displacement is used to remove the detectably labeled oligonucleotide in the removal step. In some embodiments, heat is used to dissociate the detectably labeled oligonucleotide in the removal step.

[0321] In some embodiments, the removing step comprises photobleaching.In some embodiments, photobleaching destroys a dye, such as a fluorophore, of a detectably labeled oligonucleotide.

[0322] In some embodiments, the first group and the second group of detectably labeled oligonucleotides target different sequences of each target, and the removal step after the first imaging step is optional.For example, a strategy is to target the same RNA with different DNA probes (detectably labeled DNA oligonucleotides) so that the first multiple probes can target a group of sequences on RNA, and the second multiple probes target different sequence groups on the same RNA.When the first hybridization (contact), the first multiple probes are used.They can be imaged subsequently and optionally photobleached or digested by other methods of DNA enzyme or destruction oligonucleotide or dye.The second group of probes can be hybridized and imaged, without the interference of the first group of probes.

[0323] In some embodiments, the provided methods optionally include HCR, light sheet microscopy, CLARITY, or a combination thereof. In some embodiments, the provided methods allow for direct spectral analysis of targets in tissues, organs, or organisms. In some embodiments, the organ is the brain. In some embodiments, the provided methods allow for direct imaging of transcripts in intact brains or tissues. In some embodiments, the provided methods further include HCR. In some embodiments, the provided methods further include light sheet microscopy. In some embodiments, the provided methods further include CLARITY.

[0324] The provided method provides many advantages that surpass the methods before the present invention.For example, in some embodiments, the provided method provides high throughput at a reasonable cost.In some embodiments, the provided method provides direct detection of the target without converting the target or amplifying the target.In some embodiments, the provided method enables rapid scale amplification without the need for a large amount of detectable labels.In some embodiments, the provided method can apply multiple labels to the same target and therefore increase signal strength.In some embodiments, the provided method provides a combination of the advantages.

[0325] In some embodiments, the invention provides compositions comprising a plurality of detectably labeled oligonucleotides, for example, for use in the provided methods. Exemplary compositions include, but are not limited to, those described in the exemplary method embodiments herein.

[0326] In some embodiments, the invention provides a composition comprising a plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid and is labeled with a detectable moiety, such that the composition comprises at least:

[0327] (i) a first oligonucleotide that targets a first nucleic acid and is labeled with a first detectable moiety; and

[0328] (ii) a second oligonucleotide targeted to a second nucleic acid and labeled with a second detectable moiety.

[0329] In some embodiments, the invention provides a composition comprising a plurality of detectably labeled oligonucleotides, each of which targets a transcript or a DNA locus and is labeled with a detectable moiety, such that the composition comprises at least:

[0330] (i) a first oligonucleotide that targets a first transcript or DNA locus and is labeled with a first detectable moiety; and

[0331] (ii) a second oligonucleotide that is targeted to a second transcript or DNA locus and is labeled with a second detectable moiety.

[0332] In some embodiments, the invention provides a kit comprising a plurality of detectably labeled oligonucleotides, each of which targets a transcript or a DNA locus and is labeled with a detectable moiety, such that the kit comprises at least:

[0333] (i) a first oligonucleotide that targets a first transcript or DNA locus and is labeled with a first detectable moiety;

[0334] (ii) a second oligonucleotide that is targeted to a second transcript or DNA locus and is labeled with a second detectable moiety.

[0335] (iii) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeted to the first transcript or DNA locus and labeled with the first detectable moiety, the second detectable moiety, or the third detectable moiety; and

[0336] (iv) a fourth oligonucleotide, which is optionally identical in sequence to the second oligonucleotide, which targets a second transcript or DNA locus and is labeled with a first detectable moiety, a second detectable moiety, a third detectable moiety, or a fourth detectable moiety, wherein the third oligonucleotide is labeled with a different detectable moiety than the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than the second oligonucleotide, or both.

[0337] In some embodiments, detectably labeled oligonucleotides in the composition that are targeted to the same target (transcript or DNA locus) are labeled with a moiety that provides the same detectable signal or a detectable signal that is indistinguishable in the imaging step. In some embodiments, detectably labeled oligonucleotides in the composition that are targeted to the same target are labeled with the same detectable moiety.

[0338] In some embodiments, the detectable moiety is or comprises a fluorophore. In some embodiments, the detectable moiety is a fluorophore. Exemplary fluorophores are widely known and used in the art, such as, but not limited to, fluorescein, rhodamine, Alexa Fluor, Dylight Fluor, ATTO dyes, or any analogs or derivatives thereof.

[0339] In some embodiments, the first detectably labeled oligonucleotide and the second detectably labeled oligonucleotide target different targets. In some embodiments, the first detectably labeled oligonucleotide and the second detectably labeled oligonucleotide target the same target. In some embodiments, the detectably labeled oligonucleotide in the test kit targets two or more targets, for example, transcripts and / or DNA loci. In some embodiments, the detectably labeled oligonucleotide in the test kit targets two or more transcripts. In some embodiments, the detectably labeled oligonucleotide in the test kit targets two or more DNA loci. In some embodiments, the detectably labeled oligonucleotide in the test kit targets at least 4 targets. In some embodiments, the detectably labeled oligonucleotide in the test kit targets at least 9 targets. In some embodiments, the detectably labeled oligonucleotide in the test kit targets at least 16 targets. In some embodiments, the detectably labeled oligonucleotide in the test kit targets at least 25 targets. In some embodiments, the detectably labeled oligonucleotide in the test kit targets at least 36 targets. In some embodiments, the detectably labeled oligonucleotide in the test kit targets at least 50 targets. In some embodiments, the detectably labeled oligonucleotides in the kit target at least 100 targets. In some embodiments, the detectably labeled oligonucleotides in the kit target at least 200 targets. In some embodiments, the detectably labeled oligonucleotides in the kit target at least 500 targets. In some embodiments, the detectably labeled oligonucleotides in the kit target at least 1,000 targets. In some embodiments, the detectably labeled oligonucleotides in the kit target at least 5,000 targets. In some embodiments, the detectably labeled oligonucleotides in the kit target at least 10,000 targets. In some embodiments, the detectably labeled oligonucleotides in the kit target at least 50,000 targets. In some embodiments, the detectably labeled oligonucleotides in the kit target at least 100,000 targets. In some embodiments, the detectably labeled oligonucleotides in the kit target at least 1,000,000 targets.

[0340] In some embodiments, the first oligonucleotide and the second oligonucleotide have different oligonucleotide sequences. In some embodiments, the first detectable portion and the second detectable portion are different. In some embodiments, the first detectable portion and the second detectable portion are identical.

[0341] In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 5% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 10% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 20% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 30% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 40% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 50% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 60% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 65% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 68% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 70% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 80% sequence identity. In some embodiments, the first oligonucleotide and the second oligonucleotide have less than 90% sequence identity.

[0342] In some embodiments, each oligonucleotide has less than 5% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 10% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 20% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 30% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 40% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 50% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 55% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 60% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 65% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 68% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide has less than 70% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide shares less than 80% sequence identity with any other oligonucleotide. In some embodiments, each oligonucleotide shares less than 90% sequence identity with any other oligonucleotide.

[0343] In some embodiments, the kit comprises two or more detectably labeled oligonucleotides targeting the same target. In some embodiments, 5, 10, 20, 30, 40, 50 or more detectably labeled oligonucleotides target the same target.

[0344] The detectably labeled oligonucleotide can be a variety of suitable lengths. In some embodiments, the length of the detectably labeled oligonucleotide is 15 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 16 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 17 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 18 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 19 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 20 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 21 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 22 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 23 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 24 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 25 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 26 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 27 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 28 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 29 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is 30 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 15 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 16 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 17 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 18 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 19 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 20 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 21 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 22 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 23 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 24 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 25 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 26 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 27 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 28 base pairs.In some embodiments, the length of the detectably labeled oligonucleotide is at least 29 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 30 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 35 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 40 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is at least 50 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 15-25 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 20-30 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 25-35 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 30-40 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 35-45 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 40-50 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 15-30 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 20-30 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 15-35 base pairs. In some embodiments, the length of the detectably labeled oligonucleotide is about 20-35 base pairs.

[0345] In some embodiments, a plurality of detectably labeled oligonucleotides comprise two detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise three detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise four detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise five detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise six detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise seven detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise eight detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise nine detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise ten detectable moieties.

[0346] In some embodiments, a plurality of detectably labeled oligonucleotides comprise at least two detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise at least three detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise at least four detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise at least five detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise at least six detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise at least seven detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise at least eight detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise at least nine detectable moieties. In some embodiments, a plurality of detectably labeled oligonucleotides comprise at least ten detectable moieties.

[0347] In some embodiments, the composition further comprises:

[0348] (iii) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, the third oligonucleotide targeting the first transcript or DNA locus; and

[0349] (iv) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, which targets a second transcript or DNA locus, wherein the third oligonucleotide is labeled with a detectable moiety different from that of the first oligonucleotide, or the fourth oligonucleotide is labeled with a detectable moiety different from that of the second oligonucleotide, or both.

[0350] In some embodiments, the third oligonucleotide is identical to the first oligonucleotide in sequence. In some embodiments, the third oligonucleotide comprises a sequence overlapping with the first oligonucleotide. In some embodiments, the third oligonucleotide has less than 50% sequence identity with the first oligonucleotide. In some embodiments, the third oligonucleotide has less than 40% sequence identity with the first oligonucleotide. In some embodiments, the third oligonucleotide has less than 30% sequence identity with the first oligonucleotide. In some embodiments, the third oligonucleotide has less than 20% sequence identity with the first oligonucleotide. In some embodiments, the third oligonucleotide has less than 10% sequence identity with the first oligonucleotide. In some embodiments, the third oligonucleotide has less than 5% sequence identity with the first oligonucleotide.

[0351] In some embodiments, the fourth oligonucleotide is identical to the second oligonucleotide in sequence. In some embodiments, the fourth oligonucleotide comprises a sequence overlapping with the second oligonucleotide. In some embodiments, the fourth oligonucleotide has less than 50% sequence identity with the second oligonucleotide. In some embodiments, the fourth oligonucleotide has less than 40% sequence identity with the second oligonucleotide. In some embodiments, the fourth oligonucleotide has less than 30% sequence identity with the second oligonucleotide. In some embodiments, the fourth oligonucleotide has less than 20% sequence identity with the second oligonucleotide. In some embodiments, the fourth oligonucleotide has less than 10% sequence identity with the second oligonucleotide. In some embodiments, the fourth oligonucleotide has less than 5% sequence identity with the second oligonucleotide.

[0352] In some embodiments, the third oligonucleotide is labeled with a different detectable moiety than the first oligonucleotide.In some embodiments, the fourth oligonucleotide is labeled with a different detectable moiety than the second oligonucleotide.

[0353] In some embodiments, the amount of detectably labeled oligonucleotides in a variety, composition, kit or method is predetermined. In some embodiments, the amount of 5% of detectably labeled oligonucleotides in a variety, composition, kit or method is predetermined. In some embodiments, the amount of 10% of detectably labeled oligonucleotides in a variety, composition, kit or method is predetermined. In some embodiments, the amount of 20% of detectably labeled oligonucleotides in a variety, composition, kit or method is predetermined. In some embodiments, the amount of 30% of detectably labeled oligonucleotides in a variety, composition, kit or method is predetermined. In some embodiments, the amount of 40% of detectably labeled oligonucleotides in a variety, composition, kit or method is predetermined. In some embodiments, the amount of 50% of detectably labeled oligonucleotides in a variety, composition, kit or method is predetermined. In some embodiments, the amount of 60% of detectably labeled oligonucleotides in a variety, composition, kit or method is predetermined. In some embodiments, the amount of 70% of the detectably labeled oligonucleotides in a plurality, composition, kit, or method is predetermined. In some embodiments, the amount of 80% of the detectably labeled oligonucleotides in a plurality, composition, kit, or method is predetermined. In some embodiments, the amount of 90% of the detectably labeled oligonucleotides in a plurality, composition, kit, or method is predetermined.

[0354] In some embodiments, the amount of at least 5 detectably labeled oligonucleotides in a plurality of, compositions, kits, or methods is predetermined. In some embodiments, the amount of at least 10 detectably labeled oligonucleotides in a plurality of, compositions, kits, or methods is predetermined. In some embodiments, the amount of at least 20 detectably labeled oligonucleotides in a plurality of, compositions, kits, or methods is predetermined. In some embodiments, the amount of at least 30 detectably labeled oligonucleotides in a plurality of, compositions, kits, or methods is predetermined. In some embodiments, the amount of at least 40 detectably labeled oligonucleotides in a plurality of, compositions, kits, or methods is predetermined. In some embodiments, the amount of at least 50 detectably labeled oligonucleotides in a plurality of, compositions, kits, or methods is predetermined. In some embodiments, the amount of at least 60 detectably labeled oligonucleotides in a plurality of, compositions, kits, or methods is predetermined. In some embodiments, the amount of at least 70 detectably labeled oligonucleotides in a plurality of, compositions, kits, or methods is predetermined. In some embodiments, the amount of at least 80 detectably labeled oligonucleotides in a plurality, composition, kit, or method is predetermined. In some embodiments, the amount of at least 90 detectably labeled oligonucleotides in a plurality, composition, kit, or method is predetermined. In some embodiments, the amount of at least every detectably labeled oligonucleotide in a plurality, composition, kit, or method is predetermined.

[0355] In some embodiments, two or more detectably labeled oligonucleotides are provided for a target. In some embodiments, the total amount of all detectably labeled oligonucleotides for a target is predetermined. In some embodiments, the total amount of all detectably labeled oligonucleotides for a target is predetermined, wherein the amount of each detectably labeled oligonucleotide for the target is independently and optionally predetermined. In some embodiments, the total amount of all detectably labeled oligonucleotides for each of a variety of targets is predetermined. In some embodiments, a variety of targets have at least two targets. In some embodiments, a variety of targets have at least five targets. In some embodiments, a variety of targets have at least 10 targets. In some embodiments, a variety of targets have at least 50 targets. In some embodiments, a variety of targets have at least 100 targets. In some embodiments, a variety of targets have at least 500 targets. In some embodiments, a variety of targets have at least 1,000 targets.

[0356] In some embodiments, the targets of a plurality, composition, kit, or method are predetermined. In some embodiments, at least 10 targets of a plurality, composition, kit, or method are predetermined. In some embodiments, at least 50 targets of a plurality, composition, kit, or method are predetermined. In some embodiments, at least 100 targets of a plurality, composition, kit, or method are predetermined. In some embodiments, at least 1,000 targets of a plurality, composition, kit, or method are predetermined. In some embodiments, up to F targets of a plurality, composition, kit, or method are predetermined. N The species targets are predetermined, where F is the number of detectable moieties in the species and N is the number of imaging steps.

[0357] Methods for synthesizing detectably labeled oligonucleotides are widely known and practiced in the field, for example, referring to Lubeck, E. & Cai, L. Nat. Methods 9, 743-48 (2012). Oligonucleotides are also commercially available from multiple suppliers. In some embodiments, the invention provides methods for preparing detectably labeled oligonucleotides. In some embodiments, the invention provides methods for preparing intermediate oligonucleotides. In some embodiments, the invention provides methods for preparing bridging oligonucleotides.

[0358] In some embodiments, the present invention provides a method for preparing a target nucleic acid having a first sequence, the method comprising the following steps:

[0359] 1) providing a first nucleic acid comprising a first sequence, wherein the first sequence is flanked at both ends by nicking endonuclease sites;

[0360] 2) amplifying the first nucleic acid or a portion of the first nucleic acid to provide a second nucleic acid comprising the first sequence and flanking nicking endonuclease sites; and

[0361] 3) contacting the second nucleic acid with one or more nicking endonucleases corresponding to the flanking nicking endonuclease sites.

[0362] In some embodiments, the target nucleic acid with the first sequence is single-stranded. In some embodiments, the amplification step includes polymerase chain reaction (PCR). In some embodiments, the method provided also includes the step of denaturation, wherein the second nucleic acid of double-strand is denatured and the double-strand becomes single-stranded. In some embodiments, the method provided also includes separating the nucleic acid with the first sequence. In some embodiments, before contacting with the nicking endonuclease, the second nucleic acid is optionally modified. In some embodiments, the method provided also includes marking the nucleic acid with the first sequence.

[0363] In some embodiments, two flanking endonuclease sites are identical.In some embodiments, use a kind of nicking endonuclease corresponding to identical nicking endonuclease site.In some embodiments, two flanking endonuclease sites are different.In some embodiments, use two kinds of nicking endonucleases, every kind of described two kinds of nicking endonucleases independently corresponds to a nicking endonuclease site.

[0364] In some embodiments, the oligonucleotides of the technology provided are produced from an oligonucleotide pool. In some embodiments, such pools are commercially available. In some embodiments, the initial DNA oligonucleotide pool is composed of up to 12,000 or more single-stranded sequences organized into subgroups. Every sequence is designed so that the flank of the desired sequence (e.g., probe sequence) is a nicking endonuclease site and a forward primer sequence and a reverse primer sequence. The forward primer sequence and the reverse primer sequence are specific to the subgroup to which the desired sequence belongs. Primer pairs can be used to utilize polymerase chain reaction (PCR) to amplify subgroups. The product of the PCR reaction is separated and digested by a nicking endonuclease. The time of incubation with the nicking enzyme varies based on the amount of the enzyme used and the amount of the DNA recovered. In some embodiments, the enzyme digestion of about 1 μg of DNA of about 10 units in about 1 hour. The sample is then purified and reconstituted in a buffer, such as 2x loading buffer (96% formamide / 20 mM EDTA) and water to prepare a final loading buffer (48% formamide / 10 mM EDTA) and denatured, for example, by heating to 95°C to completely denature the DNA. The denatured DNA is purified and the desired product is isolated. In some embodiments, purification and / or separation comprises electrophoresis. Fig.25 An exemplary process is illustrated in FIG.

[0365] In some embodiments, the present invention provides a method for preparing a target nucleic acid having a first sequence, the method comprising the following steps:

[0366] 1) providing a first nucleic acid comprising a first sequence or a complementary sequence thereof, wherein the first sequence or the complementary sequence thereof is flanked by at least one restriction site;

[0367] 2) amplifying the first nucleic acid or a portion of the first nucleic acid to provide a second nucleic acid, the second nucleic acid comprising the first sequence and at least one flanking restriction site; and

[0368] 3) contacting the second nucleic acid with a restriction enzyme corresponding to at least one flanking restriction site to provide a third nucleic acid comprising a recessed end;

[0369] 4) contacting the third nucleic acid with a nuclease to selectively digest the strand containing the complementary sequence (if present) while retaining the strand containing the first sequence.

[0370] In some embodiments, the first sequence or its complement is independently flanked at each end by a restriction site.

[0371] In some embodiments, the present invention provides a method for preparing a target nucleic acid having a first sequence, the method comprising the following steps:

[0372] 1) providing a first nucleic acid comprising a first sequence or its complementary sequence, wherein the first sequence or its complementary sequence is flanked by restriction sites at both ends;

[0373] 2) amplifying the first nucleic acid or a portion of the first nucleic acid to provide a second nucleic acid comprising the first sequence and flanking restriction sites; and

[0374] 3) contacting the second nucleic acid with restriction enzymes corresponding to flanking restriction sites to provide a third nucleic acid comprising recessed ends;

[0375] 4) contacting the third nucleic acid with a nuclease to selectively digest the strand containing the complementary sequence (if present) while retaining the strand containing the first sequence.

[0376] In some embodiments, the target nucleic acid with the first sequence is single-stranded. In some embodiments, the amplification step includes PCR. In some embodiments, the method provided also includes separating the nucleic acid with the first sequence. In some embodiments, before contacting with the restriction enzyme, the second nucleic acid is optionally modified. In some embodiments, before contacting with the nuclease, the third nucleic acid is optionally modified. In some embodiments, the nuclease is an exonuclease III, which preferentially degrades the chain with 3'-recessed ends, and the chain with 5'-recessed ends can be retained. In some embodiments, the restriction enzyme produces 5'-recessed ends. In some embodiments, the restriction enzyme produces 3'-recessed ends. In some embodiments, after restrictive digestion, the complementary sequence has a 3'recessed end. In some embodiments, the chain containing the complementary sequence after restrictive digestion has a 3'recessed end, and the chain containing the first sequence after restrictive digestion has a 5'recessed end. In some embodiments, the method provided also includes marking the nucleic acid with the first sequence.

[0377] In some embodiments, single-stranded oligonucleotides, such as probes or intermediate oligonucleotides for seqFISH, can be produced using nuclease digestion such as exoIII nuclease digestion. Two restriction sites on the amplification (e.g., PCR) product can be used instead of two nicking sites to flank the probe and / or aptamer sequence. In some embodiments, one restriction site leaves a 3' recessed end while the other leaves a 5' recessed end. For example, EcoRI and BamHI leave a 5' recessed end, while BmtI and PacI leave a 3' recessed end. Such restriction enzymes are widely known and used in the art. Exonuclease III preferentially degrades the 3' recessed end and retains the chain with the 5' recessed end. This provides another mechanism for generating single-stranded probes from an oligonucleotide pool using PCR and restriction nucleases.

[0378] In some embodiments, the target nucleic acid provided is DNA.In some embodiments, the target nucleic acid has the sequence identical with the first sequence.In some embodiments, the target nucleic acid is an intermediate oligonucleotide, comprising a first sequence hybridized with the target and a second sequence hybridized with the second oligonucleotide, the target such as transcript or DNA locus, the second oligonucleotide such as detectably labeled oligonucleotide.In some embodiments, the target nucleic acid is an intermediate oligonucleotide, comprising a first sequence hybridized with the target and a second sequence hybridized with the detectably labeled oligonucleotide marked by HCR.In some embodiments, the target nucleic acid is a bridging probe.

[0379] In some embodiments, the method provided is used to diagnose a disease, wherein the disease is related to an abnormal number of transcripts or DNA loci. In some embodiments, the method provided is used to select a subject for treatment. In some embodiments, the method provided is used to monitor a treatment regimen. In some embodiments, the cell in the method provided is from a subject. In some embodiments, the cell in the method provided is a mammalian cell. In some embodiments, the cell in the method provided is a human cell. In some embodiments, the cell in the method provided is from a subject. In some embodiments, the cell in the method provided is from an animal. In some embodiments, the cell in the method provided is from a human subject. In some embodiments, the cell in the method provided is separated from a human subject. In some embodiments, the cell in the method provided is from a diseased tissue or a tissue susceptible to the disease. Due to the ability to simultaneously detect and quantify a large number of targets, the method provided provides significant advantages for diagnosis, treatment monitoring, and patient stratification.

[0380] In some embodiments, the provided techniques optionally include profiling of proteins, neural activity, and / or structural arrangements. In some embodiments, the provided methods include profiling of proteins in the same sample. In some embodiments, the provided methods include profiling of neural activity in the same sample. In some embodiments, the provided methods include profiling of structural arrangements.

[0381] As exemplified herein, the provided techniques are applicable to a wide variety of samples. For example, HCR-seqFISH is effective in brain slices and SPIM can roughly detect single mRNAs in CLARITY brain slices. In some embodiments, the provided techniques can be used for target spectrum analysis in mouse models of neurodegenerative diseases or in human brains. No other techniques have been able to achieve the same quality and quantity of data before the present invention. Example

[0382] The foregoing has described certain non-limiting embodiments of the present invention. Accordingly, it should be understood that the embodiments of the present invention described herein are merely illustrative of the application of the principles of the present invention. The illustrative embodiments described in detail herein are not intended to limit the scope of the claims.

[0383] In situ profiling of nucleic acids by sequential hybridization and barcoding

[0384] As described in the non-limiting examples herein, by the provided methods, nucleic acids such as mRNA in cells are profiled by a series of contacting, imaging, and removal steps ( FIGS. 2( a) and 2( b) ). Figure 3 ). Since the transcripts are fixed in the cell, the corresponding fluorescent spots remain in place during multiple rounds of hybridization and can be aligned to read the fluorophore sequence. This sequential barcode is designed to uniquely identify mRNA.

[0385] During each round of hybridization, each transcript is targeted by a set of detectably labeled oligonucleotides (in this case, FISH probes are labeled with a single type of fluorophore). The sample is imaged and then treated with DNase I to remove the FISH probes. In subsequent rounds, the mRNA is hybridized with FISH probes having the same set of oligonucleotide sequences but now labeled with a different dye. The number of available barcodes scales to F N , where F is the number of fluorophores and N is the number of hybridization rounds. For example, with 4 dyes, 8 rounds of hybridization can cover almost the entire transcriptome (4 8 =65,536).

[0386] As an example, 12 genes were barcoded in a single yeast cell using 4 dyes and 2 rounds of hybridization (4 2=16, 4 barcodes were ignored; three cycles were performed for each hybridization). Cells were immobilized on a glass surface. DNA probes were hybridized, imaged, and subsequently removed by DNase I treatment (efficiency of 88.5 ± 11.0% (SE), Figure 4 The remaining signal is photobleached ( Figure 5 Even after 6 hybridizations, mRNA was observed to be 70.9 ± 21.8% (SE) of the original intensity ( Figure 6 It was observed that 77.9 ± 5.6% (SE) of the spots colocalized in the first two hybridizations also colocalized in the third hybridization ( Figure 7 and Figure 8 ). Quantify mRNA abundance by counting the occurrence of the corresponding barcode in cells ( Fig. 9 and Fig.10 , n = 37 cells). It was shown that mRNA can be efficiently stripped and rehybridized in mammalian cells ( Fig.11 and Fig.12 As demonstrated herein, the provided methods have many advantages over known methods prior to the present invention. For example, the provided methods are rapidly scalable; even with two dyes, the encoding capacity is in principle unlimited (2 N ). During each contact step, all available detectably labeled oligonucleotides for the probe (in this embodiment, FISH probes) that increase the brightness of the signal can be used. The readout of the provided method is also robust and enables full Z-stacks of natural samples. The provided method can take advantage of the high hybridization efficiency of detectably labeled oligonucleotides, such as FISH probes (>95% of mRNA is detected; Lubeck, E. & Cai, L. Nat. Methods 9, 743-48 (2012)). Applicants note that detectably labeled oligonucleotides such as FISH probes can also be designed to distinguish a large number of splicing isoforms, SNPs, and chromosomal loci in a single cell (Levesque, MJ & Raj, A. Nat Meth 10, 246-248 (2013)). In combination with super-resolution methods (Lubeck, E. & Cai, L. Nat. Methods 9, 743-48 (2012)), the provided method enables a large number of targets (eg, transcriptome) in complex samples such as the brain to be directly imaged with single-cell resolution.

[0387] Methods and procedures

[0388] Sample preparation: MDN1-GFP yeast cells were grown in YPD supplemented with 50 mM CaCl2 to an OD of 0.3. Cells were fixed in 1% formaldehyde 5% acetic acid for 5 minutes, rinsed 3X in buffer B and spheroplasted at 30°C for 1 hour. Cells were stored in 70% EtOH at -20°C for up to two weeks.

[0389] Prepare coverslips by sonicating 3X with alternating solutions of 1M NaOH and 100% EtOH, followed by a final round of sonication in acetone. Prepare a 2% solution of (3-aminopropyl)triethoxysilane (Sigma 440140) in acetone and immediately immerse the cleaned coverslips in the solution for two minutes. Rinse the amine-modified coverslips and store them in ultrapure water at room temperature.

[0390] Pre-treat the fixed yeast cells with 0.5U / μL of DNase I solution (Roche04716728001) at 23°C for 30 minutes. After treatment, the yeast cells are adhered to the coated coverslips by physically pressing the diluted solution of yeast between two amine-modified coverslips. The coverslips are then carefully separated (pealed apart) and immediately immersed in 1% formaldehyde solution for 2.5 minutes. After fixation, the coverslips are dried and flow cells (GraceBio Labs SA84-0.5-SecureSeal) are constructed by making the flow cells coated with adhesive adhere to the coverslips. FluoSphere365nm fluorescent beads are added to the coverslips to measure the drift of multiple hybridizations (Life F8805). The flow cells covered with sealing film are stored at 4°C.

[0391] Preparation of detectably labeled oligonucleotides: Probes were prepared according to the method in Lubeck, E. & Cai, L. Nat. Methods 9, 743-48 (2012). For each target, 24 probes were used. All 24 probes for each group of genes were coupled to one of the four dyes used: Alexa 532, 594, Cy5, and Cy7.

[0392] Hybridization: Flow cells were hybridized overnight in a hybridization buffer of 10% dextran sulfate (Sigma D8906), 10% formamide, and 2X SSC at a concentration of 2 nM / probe. After hybridization, samples were washed in 30% formamide, 0.1% Triton-X100 buffer preheated to 37°C for 10 minutes before adding to room temperature samples. Samples were washed several times with 2X SSC to remove diffused probe.

[0393] Imaging: The sample was immersed in an anti-bleaching buffer (Swoboda, M. ACS Nano 6, 6364-69 (2012)): 20 mM Tris-HCL, 50 mM NaCl, 0.8% glucose, saturated Trolox (Sigma: 53188-07-1), OD 405nm The concentration of pyranose oxidase (Sigma P4234) was 0.05 and the concentration of catalase (Sigma: 9001-05-2) was 1 / 1000.

[0394] Probe replacement: After imaging, cells were washed in DNase I buffer (Roche) and allowed to stand for 4 hours in 0.5U / μL DNase I (Roche). To inhibit DNase, cells were washed twice with 30% formamide, 0.1% Trition-X 100, 2X SSC. After DNase treatment, cells were imaged again in anti-bleach buffer to determine the DNase I probe stripping ratio. To remove the remaining probe signal, the sample was bleached with 10 seconds of excitation in all imaging channels and imaged again with standard excitation time to record the residual signal.

[0395] Rehybridization: Rehybridize the samples on the microscope according to the conditions outlined above. Cover the samples with parafilm during hybridization on the microscope to prevent evaporation.

[0396] At least six rounds of hybridization were performed on the same sample. Each round of hybridization occurred overnight under a microscope, with DNase treatment and imaging during the day. In an iterative hybridization scheme corresponding to this applied, two rounds of hybridization were used to barcode mRNA. The barcoding scheme was then repeated so that the same probes were used for hyb1 and hyb3, while another set of probes was used for hyb2 and hyb4. Colocalization between Hby1 and hyb3 gave calibration for the transcripts detected, while hyb1 and hyb2 produced barcoded data.

[0397] Data analysis: ImageJ, Python and Matlab were used for data analysis. Since the sample drifted during the experiment, the raw images were aligned using a cross-correlation-based registration method determined from the DAPI channel of each imaging position. The drift correction was then propagated to the other 4 color channels corresponding to the same position. The image was then deconvolved to reduce the overlap between adjacent FISH points. Point overlap in individual channels was rarely observed, but when the images were superimposed, points in different channels overlapped with their point spread functions (PSFs). Raw data were processed based on the iterative Lucy-Richardson algorithm (Lucy, LB The Astronomical Journal. 79, 745 (1974) and Richardson, WHJ Ort. Soc. Am. 62, 55-59 (1972)). The PSF of the microscope was evaluated by averaging the bead images (~200nm diameter) measured in the DAPI channel of the microscope. Using this measured point spread function together with the Lucy-Richardson algorithm, we performed a maximum likelihood evaluation of the fluorescence emission distribution in the FISH images after computing this process for more than 20 iterations. The output of this deconvolution method provides resolved FISH data and increases the accuracy of barcode assignment.

[0398] A local maximum function was used to identify spots corresponding to FISH signals in the image. Spots below the threshold were discarded and not used for further analysis. The value of the threshold was determined by optimizing the colocalization between hyb1 and hyb3 images hybridized with the same probe set. The maximum intensity pixel of each PSF was used as a representative of the location of that mRNA molecule.

[0399] Barcodes were automatically extracted from spots corresponding to mRNAs in hyb1 and hyb2. The algorithm calculated pairwise distances between each spot identified in hyb1 and all spots identified in hyb2. For each spot in hyb1, the nearest neighbor spot in hyb2 was identified. A barcode pair was confirmed if the distance was 0 pixel or 1 pixel and the nearest neighbor of the spot in hyb2 was also the original spot in hyb1. The requirement for symmetric nearest neighbors reduced false assignments of barcodes. To reduce false positives for cy7, a spot cy7 detected in hyb1 was required to be repeated as cy7 in hyb3.

[0400] In this non-limiting example, due to its low cost and rapid activity, the applicant uses DNase I to remove the probe. The applicant notes that any method that removes the probe from the mRNA and leaves it intact can be used for the provided barcoding method, such as, but not limited to, strand displacement (Duose, DYNucleic Acids Research, 40, 3289-3298 (2012)) and high temperature washing or formamide washing. The applicant notes that DNase I does not require redesigning the probe from a standard smFISH probe and does not interfere with the sample due to harsh washing.

[0401] In some embodiments, it is observed that the DAPI signal from dsDNA disappears quickly in a few seconds, while the smFISH probe requires a relatively long time period (tens of minutes) to be degraded. It is not intended to be limited by theory, and relative to the dsDNA cleavage rate, the probe removal efficiency of DNase I is low. Removal process is still observed in a short time.

[0402] In certain experiments, 11.5% of the fluorescent signal remained on the mRNA after DNase I treatment. The remaining signal was reduced to almost zero by bleaching. Applicants note that more complete signal and / or probe removal can be achieved before photobleaching so that more mRNA can be used for subsequent rounds of hybridization. Applicants note that photobleaching is not necessary for barcoding, but in some embodiments, it does simplify the process by removing residual signal that may give false positives in further barcoding rounds. Some of the 11.5% of residual probe bound to the mRNA may inhibit further hybridization rounds. Applicants note that since the data for the 5 hybridizations presented show a slight decrease in hybridization efficiency, the residual probe does not significantly inhibit further hybridization rounds.

[0403] Nucleic acid profiling in brain tissue

[0404] Transcriptional profiling of cells in intact brain slices is important for understanding the molecular basis of cell identity. However, prior to the present invention, quantitative profiling of transcript abundance and localization in single cells in the context of the anatomy of natural neural networks was technically difficult. The cortical somatosensory subnetwork was used as an example to illustrate the feasibility and practicality of the technology provided exemplarily, for example, using FISH in situ sequencing (seqFISH) and connectomics to analyze multiple gene profiles in different neuronal populations in different functional areas, such as those in the primary somatosensory cortex (SSp), primary somatomotor cortex (MOp), secondary somatomotor cortex (MOs), and supplementary somatosensory cortex (SSs).

[0405] As broadly described herein, in some embodiments, the present invention provides techniques for profiling gene expression in single cells via in situ "sequencing" by FISH, e.g., as described by Figure 1 2. To detect individual mRNAs, single molecule fluorescence in situ hybridization (smFISH) was used with 20mer oligonucleotide probes complementary to the mRNA sequence (Fan, Y. Braut, SA, Lin, Q., Singer, RH, Skoultchi, AI. Determination of transgenic loci by expression FISH. Genomics. 2001 Oct 2; 71(1): 66-9; Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 2006 Oct; 4(10): e309). By placing 24 such fluorophore-labeled probes on the mRNA, single transcripts in the cell become readily detectable in situ. It was shown that by smFISH, almost all detectable mRNAs were observed (Lubeck, EL Cai. Single cell systems biology by super-resolution imaging and combinatorial labeling. Nature Methods 9, 743-48 (2012)). The provided method is highly quantitative and preserves spatial information within tissue samples without physically isolating single cells or using homogenates.

[0406] In some embodiments, in order to distinguish different mRNA species, the mRNA is barcoded using sequential hybridization rounds with detectably labeled oligonucleotides such as FISH probes. During one round of hybridization, each transcript is targeted by a set of multiple probes labeled with a single type of fluorophore, for example 24 FISH probes. The sample is imaged and the FISH probes are removed by enzymatic digestion. The mRNA is then hybridized with the same FISH probes (but in some cases, now labeled with different dyes) in subsequent rounds. Since the transcripts are fixed in the cell, the fluorescent spots corresponding to a single mRNA remain in place during multiple rounds of hybridization and can be aligned to read the color sequence. Each mRNA species is therefore assigned a unique barcode. The number of each transcript in a given cell can be determined by counting the number of corresponding barcodes. Figure 1 Exemplary methods are illustrated in , 2 or 3.

[0407] The provided techniques can take advantage of the high hybridization efficiency of FISH (>95% of mRNA is detected). In some embodiments, base pair resolution is not required for identifying transcripts, although base pair resolution is achievable if desired. The number of barcodes available for the provided methods scales to F N , where F is the number of different fluorophores and N is the number of hybridization rounds. Using 5 different dyes and 3 rounds of hybridization, 125 unique nucleic acid profiles can be analyzed. 6 =15,625) almost the entire transcriptome can be covered, for example using super-resolution microscopy that resolves all transcripts in a cell. In some embodiments, fewer but still large numbers of targets, such as 100 gene multiplexes, are detected using conventional microscopy, such as conventional epifluorescence microscopy, which is simple and robust to implement.

[0408] In multiple hybridization cycles, the probe can be stripped and re-hybridized to the same mRNA (Figure 2). Many commercially available fluorophores work robustly, such as AlexaFluor 488, 532, 594, 647, 700, 750, and 790, which give at least 7 colors for barcoding. Even at the end of 6 rounds of hybridization, the probe can re-hybridize to the stripped mRNA with 70.9 ± 21.8% of the original intensity ( Figure 6 As an example, using 4 dyes and 2 rounds of hybridization, 12 barcoded genes were barcoded in a single yeast cell (4 2 =16, Figure 2, b).

[0409] Since a multiplexed image of 12 genes only occupies 5% of the optical space of each fluorescent channel, there is enough optical space in the cell to multiplex, for example, 100 genes. Figure 3 The composite image of all 4 fluorescence channels in the hybridization looks dense, but the points in each fluorescence channel are scattered. A 2D Gaussian curve can be fitted to each point to extract its centroid position and further reduce the overlap with points in other fluorescence channels. It shows that the same points are realigned to 100 nm ( Figure 8 ).

[0410] In some embodiments, 100 kinds of gene multiplexing can be carried out quickly with 3 rounds of hybridization. In some embodiments, each hybridization cycle includes about 4 hours of hybridization, about 1 hour of imaging and about 1 hour of DNA enzyme treatment and washing, and the time length can be optionally and independently changed at each time. In some embodiments, 3 rounds of hybridization cost about 18 hours. In some embodiments, the speed-limiting step is the imaging time, rather than the hybridization time, because a brain slice can be imaged while another slice hybridization on the same microscope. In some embodiments, a single microscope can multiplex 8 slices simultaneously and obtain 100 kinds of gene data to all 8 slices at the end of three hybridization cycles in 18 hours.

[0411] In some embodiments, the microscope contains 10 6 A 10mm×5mm×10μm brain slice with 10 cells can be imaged and analyzed in 35 minutes. In some embodiments, with a 20× gas eyepiece (airobjective) and a 13mm×13mm camera chip, a single field of view (FOV) on the microscope is 0.5mm×0.5mm×2μm. In some embodiments, each FOV is exposed and read at 100 milliseconds. In some embodiments, scanning the sample in XYZ and in different color channels causes a time delay of 200 milliseconds between snapshots. In some embodiments, the entire brain slice can be imaged in 2000 seconds or 35 minutes. For the 3 rounds of hybridization required for 100 gene multiplexing, the total imaging time is 105 minutes. In some embodiments, the entire mouse brain can be imaged within 30 days on a microscope. When using a multi-channel microscope, the time frame can be further shortened. In some embodiments, the provided method can image the entire mouse brain with 500 slices at a cost of less than $25,000.

[0412] The provided techniques offer a number of advantages over other methods known prior to the present invention. Among others, the provided techniques are quantitative, preserve spatial information and are inexpensively scalable to whole tissue, organ and / or organism scale.

[0413] Compare with single cell RNA-seq prior to the present invention. Unlike single cell RNA-seq or qPCR, which require single cells to be isolated and placed into a 96-well format, the provided methods such as seqFISH can scan large numbers of cells in their natural anatomical context with automated microscopy at little additional cost. Achieving the same level of throughput with a microfluidic device would be economically unfeasible and labor intensive. In some embodiments, the main expense of the provided technology is the initial expense of probe synthesis, which is offset by the fact that after the probes are synthesized, they can be used for many, e.g., 1000 to 10,000 or even more reactions.

[0414] The provided methods such as seqFISH are based on single molecule FISH and can measure low copy number transcripts with absolute quantification. The data obtained with this method are highly quantitative and enable high-quality statistical analysis. In contrast, current single-cell qPCR and RNA-seq experiments have limited quantitative capabilities due to bias from reverse transcription (RT) errors and PCR amplification errors.

[0415] Compared with other in situ sequencing methods before the present invention. A major advantage of the smFISH method is that almost all mRNAs targeted can be observed. It has been determined that the efficiency of each FISH probe bound to mRNA is 50%-60% (Lubeck, E. & Cai, L. Nat. Methods 9, 743-48 (2012); Levesque, MJ & Raj, A. Nat Meth 10, 246-248 (2013)). Targeting multiple, for example, 24 to 48 probes per mRNA ensures that at least 10 probes hybridize on almost every mRNA, providing a good signal in a non-specific background. Directly detecting mRNA with FISH probes is highly specific and ensures that all transcripts are detected.

[0416] In contrast, many other in situ sequencing methods utilize enzymatic reactions to convert mRNA into DNA templates, rather than directly targeting mRNA, before detecting the DNA template by sequencing reactions. However, the conversion process of mRNA to DNA is highly inefficient, and only a small proportion of RNA is converted and detected. An exemplary major disadvantage of low efficiency (estimated to be 1% for reverse transcription (RT) and 10% for padlock ligation (PLA)) is that it can introduce significant noise and bias in gene expression measurements.

[0417] Assuming a typical cell size of (10-20 μm 3 ), there are about 25,000 diffraction-limited spots in the cell. In some embodiments, this is a real estate available for transcript detection in single cells. In seqFISH, a selected set of genes, such as transcription factors (TFs) and cell adhesion molecules (CAMs), can be imaged and quantified with high precision. If target genes with an average copy number of 100 transcripts per gene are selected, highly quantitative profiling experiments of 100-200 genes can be performed. In contrast, for many other in situ sequencing methods, the majority of the real estate is used to sequence ribosomal RNA and housekeeping genes; genes of interest such as those specific to neural cell identity are severely under-represented and difficult to detect.

[0418] In some embodiments, the provided methods use hybridization chain reaction (HCR) (Choi, et al., Programmable in situ amplification for multiplexed imaging of mRNA expression Nature Biotechnol, 28, 1208-1212, (2010)) to amplify FISH signals, which allows large FOV imaging with a 20X gas eyepiece, while retaining the high detection efficiency of smFISH.

[0419] Compared with the methods for super-resolution barcoding of multiplexed RNA before the present invention. In some embodiments, compared with the methods for barcoding mRNA profiles by smFISH before the present invention (Femino et al., Visualization of single RNA transcripts in situ. Science. April 24, 1998; 280(5363):585-90; Kosman et al., Multiplex detection of RNA expression in Drosophila embryos. Science. August 6, 2004; 305(5685):846; Levsky et al., Single-cell gene expression profiling. Science. August 2, 2002; 297(5582):836-40; Lubeck et al., Single cell systems biology by super-resolution imaging and combinatorial labeling. Nature Methods 9, 743-48 (2012); and Levesque et al., Nat Meth 10, 246-248 (2013)), the provided method has many advantages over the previous invention, in which probes for specific mRNAs are divided into subsets labeled with different dyes. Among other things, the provided technology does not require many different fluorophores to scale up; even with two dyes, the encoding capacity is huge, and repeated barcodes (e.g., red-red-red) can be used. In comparison, barcoding of RNA spectra before the present invention was limited in the number of barcodes that could be generated (~30). In the provided method, during each round of hybridization, all detectably labeled oligonucleotides such as FISH probes for transcripts can be used immediately, rather than dividing the probes into subsets. Among other things, due to the stronger signal on each mRNA, the provided technology provides improved robustness of barcode reading. Compared to the methods before the present invention, since each mRNA can have fewer colors, in some embodiments, a single color during each round of hybridization, rather than at least three colors in the barcoding scheme before the present invention, the density of targets in the image is lower. Before super-resolution microscopy is necessary, lower image density can greatly simplify data analysis and allow more genes to be multiplexed, if desired. Applicants note that in the provided embodiments, certain spectral barcoding methods, probes, and / or super-resolution microscopy can be used and can be useful embodiments.In some embodiments, to profile the transcriptome using provided techniques such as seqFISH, super-resolution microscopy is used to resolve millions of transcripts in a cell.

[0420] In addition to the spectral analysis of transcripts, the provided technology can distinguish multiple alternative splicing choices and RNA editing for the same mRNA molecule. Since sequencing reads are usually too short to be associated with exons within the same transcript, alternative splicing isoforms are difficult to detect by sequencing methods. The provided methods such as seqFISH allow direct visualization of all splicing isoforms within individual cells in brain slices. Similarly, the smFISH method for detecting single nucleotide polymorphisms (SNPs) can be applied to seqFISH to image edited transcripts in neurons or other cell types.

[0421] In some embodiments, the provided technology provides efficient and cost-effective pipelines for the following: in situ profiling of somatic motor neurons in the cortex by sequential FISH (seqFISH), and integrate seqFISH and connectomics to identify combined molecular markers corresponding to cell identity.

[0422] In situ quantification of gene expression mapping in the brain

[0423] Light sheet microscopy is applied to directly image CLARITY-clarified brain slices. In some embodiments, one mouse brain is mapped in 1 month per machine. In some embodiments, one mouse brain is mapped in one week using 4-5 machines.

[0424] Amplification: Amplification of FISH signals allows for large FOV imaging of brain sections using a 20X low NA objective. In some embodiments, the provided methods use detectably labeled oligonucleotides labeled by hybridization chain reaction (HCR) (Choi et al., 2010) to increase the signal-to-background ratio and / or preserve the specificity and multiplexing capabilities of the FISH method. With this approach, nucleic acid probes complementary to the mRNA target trigger a chain reaction in which metastable fluorescently labeled nucleic acid hairpins self-assemble into tethered fluorescent amplification polymers. Using orthogonal HCR amplifiers carrying spectrally distinct fluorophores, all channels can be amplified simultaneously in situ.

[0425] In some embodiments, the detectably labeled oligonucleotide with HCR (HCR probe) comprises a 20-nt region complementary to the target mRNA, plus a 40-nt HCR initiator. The hybridization of the probe is carried out under the stringency of 10% formamide, followed by an amplification step under permissive conditions. Conditions such as hairpin concentrations can be optimized to achieve optimal results; the applicant points out that in some cases, higher concentrations of hairpins increase the reaction rate. Each HCR probe can be amplified to a diffraction-limited spot. In some embodiments, within the scale of the diffraction-limited spot, the FISH signal is amplified by about 10-20 times. The spot brightness can be further enhanced while maintaining the diffraction-limited spot scale by, for example, incorporating multiple HCR initiators in each probe and / or labeling each HCR amplification hairpin with multiple fluorophores.

[0426] In brain sections, HCR-amplified signals were observed directly from mRNA. When targeting the same mRNA, the HCR probe colocalized with the smFISH dots at a rate of 90%, but was 10-20 times brighter ( Fig.14 This allows the HCR probe to be easily detected beyond the autofluorescence of the brain ( Fig.13 ). The high localization ratio demonstrated that HCR was as specific as smFISH and most transcripts were detected.

[0427] HCR probes are easily stripped and rehybridized and can be fully integrated with the seqFISH protocol described herein. Fig.15 The same gene targeted by HCR in two different rounds of hybridization in brain sections is shown. The good co-localization between the two hybridizations shows that HCR-seqFISH robustly barcodes mRNA in the brain.

[0428] HCR scheme and smFISH hybridization spend the same time range and do not increase the cycle time of determination.The initial hybridization step in HCR is similar to smFISH in time, and the second amplification step occurs in 30 minutes and 1 hour.The optional method of making RNA probe hybridize with transcript and optionally using optional hairpin type to amplify signal can further reduce the cycle time.In some embodiments, HCR eliminates the demand for purchasing oligonucleotide probes labeled with amines.Among others, HCR can potentially reduce the reagent cost of about half, for example, to $10,000 per brain.

[0429] Automation. Automation of both hardware and software can be applied to effectively scale up, for example, mapping 100 genes and / or reducing human labor and / or errors. In some embodiments, the key parts of the technology are integrated to produce a pipeline and / or optimized workflow for imaging of tissues and / or organs, such as brain slices. Among others, automated fluidics, image acquisition and / or integrated analysis can be independently and optionally combined with fast hybridization cycle time and imaging time.

[0430] Hardware. In some embodiments, the automated system requires minimal intervention from the user and can automatically perform image acquisition after the user has set up the experiment. In some embodiments, each sequencer consists of or includes an automated epifluorescence microscope for imaging and an automated fluidic system for continuous hybridization. In some embodiments, compressed air is used to push reagents into a 1 cm×1 cm hole with cells and tissues fixed on a bottom coverslip. Not intending to be limited by theory, the applicant points out that in some embodiments, due to the high viscosity of the hybridization buffer, the compressed air drive system eliminates dead volume and can also be precisely controlled to deliver reagents of a defined volume. In some embodiments, a separate vacuum line is used for a clean room. In some embodiments, the workflow of the provided scheme is similar to existing DNA sequencers known in the art at the time of the present invention.

[0431] In some embodiments, during each hybridization cycle, the machine automatically hybridizes the sample with the probe, washes with buffer to remove excess probe and / or scans the brain slice for imaging. In some embodiments, wherein DNA enzyme is used in the removal step, the DNA enzyme flows in to remove the probe after imaging. After thorough washing, another round of hybridization can be performed subsequently. During the hybridization time, the microscope moves to different positions on the stage to image another brain slice that has been hybridized and washed. In this way, most of the time the camera is collecting images while other samples on the stage are hybridizing.

[0432] Software. In some embodiments, software is used, for example, to automate the control process and analyze data. In some embodiments, code is written in Micromanager (free software supported by the National Institute of Health) to control the microscope and fluidic elements. In some embodiments, valves, stages, light sources, cameras, and / or microscopes are controlled by Micromanager.

[0433] In some embodiments, compressed sensing is used for dense images (Zhu et al., Faster STORM using compressed sensing. Nat. Methods. 2012, 9 (7): 721-3) and deconvolution methods are used to distinguish points in dense clusters. In some embodiments, improvements in image analysis improve the multiplexing capabilities of the provided methods, such as seqFISH (e.g., about 4-5 times more than 100 gene multiplexing). In some embodiments, efficiency is improved in a similar manner from the Illumina GAII sequencer to the HiSeq instrument, where the use of image processing methods to analyze densely packed clusters on the sequencing chip increases throughput. In some embodiments, data acquisition and analysis are integrated into a user-friendly package.

[0434] In some embodiments, the technology provided provides a software package for data analysis. In some embodiments, the technology provided provides a software package for analyzing data in Python and Matlab. The image of the technology provided can be of various sizes, and can be optionally optimized if desired. In some embodiments, each FOV is 6 megapixels with 14 bit depth, corresponding to 1.5MB of data for each image. In some embodiments, each operation generates about 100GB of data. In some embodiments, the technology provided provides a method for data processing and / or alleviating data blocking (logjam). In some embodiments, data blocking is alleviated by the following: splitting out points from each image, fitting it with a 2-dimensional Gaussian distribution, and recording the center position of the fitting point (fits). In some embodiments, the technology provided saves computer space by discarding the original image and preserving the processed data.

[0435] Light sheet illumination microscopy of brain slices clarified with CLARITY. In some embodiments, the technology provided provides a method for imaging tissues, organs and / or organisms. In some embodiments, the technology provided provides a method for measuring thick tissues or organs. In some embodiments, thick tissues or organs have a thickness of about 100 μm or greater than 100 μm. In some embodiments, the technology provided retains long-range projections and morphology beyond a single cell. In some embodiments, light sheet illumination microscopy is used to measure thick tissues or organs. In some embodiments, tissues, organs and / or organisms are clarified by CLARITY (Chung et al., Structural and molecular interrogation of intact biological systems, Nature, 2013, doi: 10.1038 / nature12107).

[0436] In some embodiments, light sheet microscopy (also known as selective plane illumination microscopy (SPIM)) is applied to CLARITY-clarified brain tissue in order to image thicker brain slices (>100 μm) that better preserve long-range projections and morphology. In some embodiments, the CLARITY method renders the brain transparent for visualization and identification of neuronal components and their molecular identities. In some embodiments, CLARITY renders brain tissue optically transparent and polymer-permeable by removing light-scattering lipids, which are replaced by porous hydrogels to preserve the morphology of brain tissue so that studies can be conducted without thinning the brain, which enables visualization of neurons of interest and their long-range synaptic connections. Without intending to be limited by theory, applicants note that the provided technology can use thicker tissue and allow for more accurate reconstruction of individual neurons or 3D neural network transcriptomes compared to FISH previously performed in culture or thin slices prior to the present invention. Fig.16 A successful, validated Clarity-based protocol for preparing optically clear thick sections compatible with FISH staining is illustrated: (1) 100-micron coronal brain sections in 2-mL Eppendorf tubes were incubated in 1.5 mL of 4% acrylamide, 2% formaldehyde, 0.25% thermal initiator, 1× PBS at 4 degrees overnight; (2) nitrogen was bubbled through the hydrogel solution for 10 seconds; (3) the degassed samples were incubated at 42 degrees for 2 hours to allow polymerization; (4) the samples were washed three times in PBS and incubated in 10% SDS, 1× PBS at 37 degrees for 4 hours to allow clarity; and (5) the samples were washed three times in PBS and used immediately for seqFISH.

[0437] In some embodiments, the provided technology provides a method for minimizing background or avoiding blurred background. In some embodiments, the provided technology utilizes imaging technology that minimizes background or avoids blurred background. In some embodiments, SPIM is used for thicker slices with higher blurred background. In some embodiments, although the confocal microscope can reject such background, it scans slowly and photobleaches the upper layers of the sample when imaging the lower layers. In some embodiments, only the imaged layer is illuminated by the excitation light in SPIM. In some embodiments, in the SPIM setting for the provided technology, two objective lenses placed perpendicular to each other are suspended about 55° above the sample. In some embodiments, the light sheet is produced using a cylindrical lens that focuses an optical axis of the light within an effective width or FOV of about 10 μm high and about 200 μm.

[0438] In some embodiments, the present invention provides a microscope setup for the provided methods. In some embodiments, the present invention provides a light sheet microscope in which the sample is illuminated from the side. In some embodiments, the light sheet is parallel to the sample stage. In some embodiments, the light sheet is perpendicular to the detection objective. Fig.17 An exemplary setup of a light sheet illumination microscope is illustrated in FIG. By refitting two reflectors and a cylindrical lens, a plane of a light sheet is generated and illuminates the sample from the side and perpendicular to the detection objective (middle). The bottom reflector is connected to the cylindrical lens and is directly mounted on the same base of the objective. For this configuration, the objective moves synchronously with the illumination sheet, allowing the sample to be scanned along the z-axis (right, top). The right (bottom) figure also shows that the sample is placed in the hybridization chamber and imaged by the air objective below.

[0439] As in Fig.18 As shown in the figure, a SPIM image was obtained of a 100 μm brain section clarified with CLARITY and hybridized with an HCR probe for β-actin. 200 optical sections with 0.5 μm intervals were used to generate the reconstruction. A 20× water immersion objective was used to observe the clear HCR signal. β-actin mRNA is highly expressed, accounting for the large number of spots in the cell body. However, clear diffraction-limited spots were also observed in the axons.

[0440] In some embodiments, the HCR-seqFISH protocol for CLARITY-cleared brains and SPIM microscopy can be modified. 100 μm sections were effectively hybridized within 4-5 hours, indicating that detectably labeled oligonucleotide probes can diffuse easily in 100 μm thick but clear sections. In addition, DNase also diffuses easily to strip HCR signals from sections ( Fig.15 In some embodiments, the provided technology provides detectably labeled oligonucleotides, such as FISH and HCR probes, that are smaller than the antibodies that one of ordinary skill in the art would routinely use to diffuse into 1 mm thick coronal sections, and provides for profiling of targets across an entire tissue or organ, e.g., seqFISH on a CLARITY clarified whole brain.

[0441] In some embodiments, the technology provided provides the geometry of microscopy. In some embodiments, the technology provided provides an optional geometry of SPIM so that thick brain slices (>100 μm) and potentially the entire CLARITY-clarified brain can be mounted on an epifluorescence microscope with a long working distance objective. In some embodiments, the light sheet is generated perpendicular to the imaging axis and spans 200-300 μm to divide the sample mounted on the microscope at a certain inclination with a 10 μm wide division. In some embodiments, the geometry provided allows the flow chamber and automated design transfer of the development to be guided. In some embodiments, the fiducial markers in the brain slices are used to record continuous slices. In some embodiments, before dividing, a nanoscale ruler is injected into the brain, allowing good recording between different partitions.

[0442] Speed. In some embodiments, the imaging speed limits the final throughput. In some embodiments, the HCR amplification provided provides more than enough photons for imaging, and a less expensive camera can be used to image the sample. In some embodiments, the light from the collection objective can be divided into multiple, for example, 6 different beams (e.g., 5 fluorescences and 1 DAPI) with imaging plane dichroism and filters. This greatly increases the throughput of the in situ "sequencer", so that the entire brain can be completed within 1 week on a single microscope.

[0443] Target gene selection. In some embodiments, the invention provides techniques for selecting and imaging a set of targets, such as a set of transcripts and / or DNA loci (e.g., a set of 100 targets as exemplified). In some embodiments, target genes are selected from an in situ database from the Allen brain atlas (ABA). Multiple criteria can be used to select genes of interest, e.g., those genes in cortical regions that may represent cell identity. The selection of the best set of genes from the standard calculation of overlap is well known (2. Alon, N; Moshkovitz, Dana; Safra, Shmuel (2006), "Algorithmic construction of sets for k-restrictions", ACM Trans. Algorithms (ACM) 2(2): 153-177, ISSN 1549-6325; 8. Cormen, TH.; Leiserson, Charles E.; Rivest, Ronald L.; Stein, Clifford (2001), Introduction to Algorithms, Cambridge, Mass.: MIT Press and McGraw-Hill, pp. 1033-1038, ISBN 0-262-03293-7; 12. Feige, U (1998), "A threshold of In n for approximating set cover", Journal of the ACM (ACM) 45 (4): 634-652,, ISSN 0004-5411). In some embodiments, set-cover-heuristics (Pe'er, 2002) is used to select the following genes: 1. Genes known to define cell subtypes; 2. Genes that exhibit a "salt and pepper" expression pattern in ABA; 3. Genes belonging to gene families such as transcription factors, ion channels, GPCRs, and neurotropin; and 4. Genes that were eliminated from RNAseq experiments on cortical samples. For example, SLC1A3 identifies glial cells while SLC6A1 identifies inhibitory neurons, and SLC17A7 identifies excitatory neurons. In some embodiments, genes with heterogeneous expression patterns such as PVALB, SST, and CALB identify a subgroup of inhibitory neurons. An exemplary group of 100 genes is shown below:

[0444]

[0445]

[0446]

[0447]

[0448] seqFISH is integrated with protein detection, organelle markers, and activity measurements. In some embodiments, the provided techniques, such as seqFISH, allow for multiplexed in situ analysis of RNA and proteins, neural activity, and structural arrangements in the same sample at single-cell resolution. In another round of hybridization, antibodies to specific targets can be hybridized to the sample. In some embodiments, the provided methods optionally include an immunostaining step. In some embodiments, multiple antibodies are used to detect many protein targets in sequential hybridization rounds (Schubert W et al. Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat Biotechnol (2006) 24 (10): 1270-1278). Applicants note that there is a higher protein abundance in cells that exceeds mRNA by as much as about 100-1000 times or more. Targeted proteins can identify organelles such as mitochondria, ER, transport vesicles, cytoskeleton, and synaptic connections. For example, MAP2 antibodies can be used to identify cell boundaries to help demarcate axons and dendrites.

[0449] Live imaging of brain sections can be performed on epifluorescence and light sheet illumination microscopes prior to transcriptional profiling by the provided methods (eg, seqFISH). In brain slices, calcium (Nakai J, Ohkura M, Imoto K (February 2001). "A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein". Nat. Biotechnol. 19(2): 137-41; Akerboom et al., "Optimization of a GCaMP calcium indicator for neural activity imaging." J Neurosci. 2012 Oct 3; 32(40): 13819-40; Stosiek et al., "In vivo two-photoncalcium imaging of neuronal networks." Proceedings of the National Academy of Sciences 100(12): 7319) and voltage sensors (Cohen, et al., "Optical Measurement of Membrane Potential" in Reviews of Physiology." Biochemistry and Pharmacalogy, Vol. 83, pp. 35-88, 1978 (June); Mutoh et al., Genetically Engineered Fluorescent Proteins. VoltageReporters ACS Chem Neurosci. 2012 Aug 15;3(8):585-592; Peterka et al., Imaging voltage in neurons. Neuron. 2011 Jan 3;69(l):9-21) can be imaged. SPIM allows efficient and rapid imaging of these sensors in brain slices. Brain slices are mounted on a microscope and the provided protocols, such as the seqFISH protocol, can be performed with automated fluidics. In some embodiments, in addition to live measurements, mRNA of activity-dependent immediate early genes (IEGs) is detected as a measure of overall neural activity in neurons. For example, CamKII and cFos are easily detected in neurons with heterogeneous expression levels; they can be incorporated into groups of genes separately according to abundance, for example, an exemplary 100 gene multiplex, or FISH can be performed in additional cycles.

[0450] Integrating connectomics and seqFISH to characterize the molecular identities of distinct neurons within distinct somatosensory motor neural networks.

[0451] The provided techniques are used to systematically characterize the molecular identities of different neuronal populations within somatic sensorimotor neural networks. In some embodiments, neuronal populations within the same functional subnetwork may share a common set of marker genes, but also have heterogeneous expression of other genes that define identity at the cellular level. In some embodiments, cells in different subnetworks are more distinct in their expression patterns. Exemplary cortical-cortical somatic subnetworks, each of which controls a basic type of sensorimotor function, are: (1) orofaciopharyngeal, for eating; (2) upper limbs, for reaching and grasping; (3) lower limbs, for movement; and (4) whiskers, for rhythmic whisker movements. In some embodiments, the provided techniques provide novel and rigorous methods for characterizing the molecular identities of cortical neurons with different neural networks and provide invaluable information for understanding the genetic circuits that underlie the wiring diagram of the mammalian brain.

[0452] Using a series of neural pathways, digital cortical connectivity maps (atlases) can be generated to display the raw images of tract tracing studies. Cortical-cortical connectivity maps can be generated by graphically reconstructing pathways to facilitate the analysis of large-scale data. Based on intracortical connections, four different cortical-cortical somatic subnetworks can be established, each of which controls basic types of sensorimotor functions. Each of these subnetworks contains 4-5 different functional domains in the primary somatosensory (SSp) cortical area, the primary somatomotor (MOp) cortical area, the secondary somatomotor (MOs) cortical area, and the supplementary somatomotor (SSs) cortical area, which are further subdivided according to the strength of their connections with other somatosensory motor areas corresponding to specific somatic subdivisions. In some embodiments, the orofacial pharyngeal subnetwork includes five major nodes: (1) SSp oral and nasal domains (SSp-m / n); (2) MOp orofacial domain (Mop-orf); (3) MOs rostrodorsolateral domain (MOs-rdl); (4) SSp barrel field anterolateral domain (SSp-bfd.al); and (5) SSs rostrodorsal and caudal ventral domain (SSs-r&cv). In some embodiments, the four major nodes of the upper limb subnetwork include (1) SSp upper limb (SSp-ul); (2) Mop-ul; (3) rostrodorsal MOs (MOs-rd); and (4) caudodorsal SSs (SSs-CD). In some embodiments, the lower limb / trunk subnetwork includes the SSp lower limb / trunk region (SSp-ll / tr), MOp-ll / tr, and rostrodorsomedial MOs (MOs-rdm) ( Fig.19 In some embodiments, the whisker subnetwork includes the caudomedial SSp-bfd (SSp-bfd.cm), corresponding to the MOp-w of the whisker primary motor cortex (vM1), and the caudal dorsal SSs (SSs-cd; Fig.19 ). Exemplary data are described by the Mouse Connectome Project (www.mouseconnectome.org).

[0453] In order to determine the molecular identity of different neuronal populations in each of these somatic sensorimotor subnetworks, multiple fluorescent retrograde tracers are used to label neurons, and the provided techniques such as seqFISH can be applied to determine the gene expression profile of the retrogradely labeled population at single-cell resolution. In order to label neuronal populations, multiple (e.g., five) retrograde tracers are injected into five major targets of one of the main nodes of each sensorimotor subnetwork in the same animal (tracer information is as follows). For example, in one animal, the circuit tracer is injected into two of the main cortical nodes (SSp-bfd.al and SSs-r&cv) and three of the subcortical nodes (caudoputamenventrolateral domain, CP-vl; ventroposterior medial nucleus of the thalamus, VPM; and ventral trigeminal spinal nucleus, SPV) of the oral-facial pharyngeal subnetwork. This simultaneously backgrounds (back) labeling of five different neurons in all other nodes of the oral-facial pharyngeal subnetwork. In this embodiment, the labeled neurons are in the SSSp-m / n domain and in the Mop-oro domain.

[0454] On the other hand, the tracer can be injected into four different SSp somatic partitions (i.e., SSp-m / n, SSp-ul, SSp-ll / tr, and SSp-bfd.cm), each of which belongs to a different somatic subnetwork. This simultaneously labels different neuronal populations in the cortical areas associated with the different subnetworks. In this case, for example, background labeled neurons can be observed in the MOp domains associated with each subnetwork, i.e., Mop-orf, Mop-ul, Mop-ll / tr, and Mop-w. The injection strategy applied to all major nodes and subcortical targets of each of the four somatic sensorimotor subnetworks labels different neuronal populations for each subnetwork.

[0455] After the tracer injection (e.g., one week after the tracer injection), the animals were sacrificed, and their brains were harvested and sectioned coronally at a thickness of 20 μm or 100 μm for seqFISH analysis of background labeled neurons. Genes were pre-selected for profile analysis using, for example, the online digital gene expression database of the Allen BrainAtlas project (www.Brian-Map.org) (Lein et al., 2007 Genome-wide atlas of gene expression in the adult mouse brain. Nature, 11; 445(7124): 168-76), such as exemplary about 100 genes enriched in somatosensory motor cortex areas (SSp, Mop, MOs, SSs).

[0456] Injection strategy and post-injection treatment. Three hundred 4-week-old C57B1 / 6 male mice were used. In one animal, five fluorescent retrograde tracers were injected as described above into different nodes within the same somatosensory motor subnetwork or into different nodes of different somatosensory subnetworks ( Fig.19 ). The tracers are fluorogold (FG, yellow), cholera toxin b conjugated to 488 or 647 (CTb-488 [green], CTb-647 [pink]), Red retrobeads (RR, red), and wheat germ agglutinin conjugated to 655 (WGA-Qdot655, white). Since Qdot655 has a different excitation wavelength than CTb647, Qdot655 can be captured into different channels and pseudo-colored with a unique colorimetry. The tracers are injected (iontophoretically or by pressure injection) via stereotaxic surgery. The details of the surgery and perfusion are described, for example, in Hintiryan et al., Comprehensive connectivity of the mouse main olfactory bulb: analysis and online digital atlas. Front Neuroanat. 2012 Aug 7; 6:30. eCollection 2012. In some embodiments, two paired mice are injected with the same tracer used in exactly the same coordinates. One of the animals was used to confirm the location of the labeled cells and injection sites, while the other was subjected to the provided seqFISH method, for example. The animals were perfused after the tracer was transported, and the brain was coronally cut into slices of 50 μm thickness and collected in four series. One of the four series of slices was counterstained with fluorescent Nissl staining solution (NeuroTraceBlue [NTB]), mounted on a slide, and imaged using an Olympus VS120 virtual microscopy system. In some embodiments, Nissl staining provides a cytostructural background for visualizing the precise anatomical position of background labeled cells. These images are processed by an information pipeline so that each individual image is faithfully aligned to the corresponding level of its Allen Reference Atlas (ARA). The Nissl staining, together with the provided information tools, enables the automatic and accurate counting of the approximate number of neuronal populations labeled by each tracer in each ROI (in this case, different domains of the somatosensory motor area). The distribution pattern is automatically plotted to the corresponding map level to generate its connection map.

[0457] Paired mice were killed at the same time, and their brains were sliced ​​at a thickness of 20 μm or 100 μm for seqFISH analysis. These slices were first imaged under a 20× (or 10×) objective to show neurons labeled with different tracer backgrounds. For an exemplary set of 100 genes, brain slices across all coronal levels containing the somatosensory motor area were used to perform seqFISH. This method shows gene expression in neurons labeled by each tracer. All images were first analyzed for the gene expression profile of neurons labeled by each individual tracer. Each slice was aligned back to the closest matching slice of its paired brain so that slices from the paired brain at approximately the same coronal level can be displayed side by side in the connectome window. In this way, in the closest matching anatomical background of different neuronal populations, their molecular profiles are displayed. In some embodiments, in each retrogradely labeled neuronal population, gene expression profiles are associated.

[0458] Results. In some embodiments, different retrogradely labeled neuron populations in different somatosensory motor areas show different transcriptome profiles; Even the neuron populations in the same domain (e.g., SSp-m) labeled with different tracers show gene expression profiles different from its neighboring neurons with different connection profiles. In some embodiments, different neuron populations in different somatosensory motor nodes (e.g., SSp-m, MOp-orf, SSp-bfd.al and SSs-r) in the same subnetwork share common network-specific genes, while neuron populations in different neural networks (e.g., oral-facial-pharyngeal and lower limb / trunk subnetworks) show very different transcriptome profiles. In some embodiments, for those neurons in different cortical areas and different layers, identification regions (e.g., SSp or MOp) or layers (different layers) specific genes.

[0459] As exemplified, the provided technology provides a unique combination of fluorescent beam tracing and seqFISH technology to characterize the molecular identity of connectivity-based neuronal populations (cell types) within different somatosensory motor networks with subneuronal resolution and faithful anatomical context. See, e.g., Figure 3 and Fig. 9 , are exemplary results. In some embodiments, the provided techniques include measuring other parameters in parallel (i.e., antibody and organelle staining, and IEG expression levels), and can be applied to the entire neocortex or brain.

[0460] High-throughput pipelines and informatics tools for online analysis and display of data. In some embodiments, the technology provided provides for analyzing and displaying data high-throughput pipelines and informatics tools online through, for example, publicly accessible databases, such as www.MouseConnectome.org. In some embodiments, the technology provided provides integration with the Mouse Connectime Project, and the wide range of research of the Mouse Connectime Project and the use of multi-fluorescence imaging make it a valuable tool among the connectome community and are very suitable for long-range connection research in the mouse brain. For example, it provides an online visualization tool, which allows the user to visualize the pathway of multi-fluorescence labels on the cell structure background of itself and on the corresponding ARA atlas level. In some embodiments, to faithfully associate seqFISH information with its corresponding retrogradely labeled somatic cell compact, labeled cell bodies are discretely segmented from tissue background and from images of the same slice, but taken in different rounds (e.g., first for imaging retrograde tracers, then for different mRNAs in seqFISH), and spatially indexed by their coordinates relative to fixed reference points on anatomical landmarks in the slice and / or tissue. In some embodiments, to associate data with stereotaxic coordinates defined in the ARA, the present invention provides a novel registration pipeline that greatly increases registration accuracy (i.e., warping each scanned microscopic image to the shape of the corresponding level of the ARA) and image segmentation that automatically and accurately enumerates fluorescently labeled neurons in a given ROI (e.g., SSp-m, MOp-11). In some embodiments, the provided techniques collectively allow labeling within the brain and seqFISH data from multiple tracers, and across multiple brains, to be collated into a single anatomical framework for visualization and annotation purposes.

[0461] In some embodiments, the images are registered at the level of the corresponding Allen Reference Atlas (Dong, HW (2008). The Allen Reference Atlas: A Digital Color Brain Atlas of C57BL / 6J Male Mouse, John Wiley & Sons). The deformation matrix generated by the registration process is applied to the original resolution image to obtain a high-resolution warped image. After registration and registration refinement, Fluorescent Nissl staining is converted to bright field images. Next, each channel of each image is adjusted for brightness and contrast to maximize label visibility and quality in tools such as iConnectome. After correction (i.e., skewness, angle) and JPEG2000 file format conversion, images can be published to the iConnectome viewer ( www.mouseconnectome.org ).

[0462] FISH visualization tool. In some embodiments, all connection data generated are processed by MCP informatics pipeline and displayed online by new iConnectome FISH viewer (www.mouseconnectome.org). Different from the iConnectome viewer that displays two anterograde (PHAL and BDA) marks and two retrograde (FG and CTb) marks, iConnectome FISH can display up to five different neuron populations with retrograde fluorescent dyes. As mentioned above, each group of injections can be given to a pair of mice. One can be processed according to conventional MCP pipeline and displayed in iConnectome FISH viewer to present multiple fluorescently labeled neuron populations in the cell structure background of Nissl staining of multiple fluorescently labeled neuron populations themselves and in their corresponding ARA levels. These can provide accurate anatomical information for each fluorescently labeled neuron population across the entire brain. After seqFISH, the brain slices from its paired partner are aligned to the closest ARA level to which its paired partner is aligned and can be displayed side by side in different windows. The list of genes expressed in neurons is listed on the side panel. After clicking on a gene, fluorescently labeled neurons expressing that gene can light up to indicate where it is expressed. This provides a practical way to visualize the molecular identity of a neuronal population within a global context that connects the background and anatomy.

[0463] In some embodiments, a corresponding database is developed that allows users to analyze these data and associate neural connections with their molecular identities. The informatics tool is based on a database that uses genetic barcoding to store information related to each retrogradely labeled neuronal population (e.g., cell number, anatomical location). The database can help users identify the corresponding genetic barcodes of neurons within the same neural network or different neural networks.

[0464] Mapping the whole brain. In some embodiments, provided techniques have sufficient sensitivity, selectivity, automation, and / or spatiotemporal resolution at the single neuron level for high-throughput analysis of gene expression in retrogradely labeled neurons throughout the brain.

[0465] Additional Exemplary Methods of Removal Step

[0466] In some embodiments, the present invention provides various methods for removing detectably labeled oligonucleotides from a target. In some embodiments, exonuclease III (ExoIII) is used to remove detectably labeled oligonucleotides. Fig.21 An exemplary method of HCR rehybridization using Exo III is shown. Fig.21 In the process, Exo III digests the bridging strand and the HCR polymer, leaving the intermediate oligonucleotide intact for hybridization with the new bridging strand. Fig.21 (b) shows exemplary data using detectably labeled oligonucleotides targeting the β-actin (Actb) transcript in T3T mouse fibroblasts. The left image shows initial hybridization and amplification of the Actb transcript using Alexa 488 dye. The middle image shows complete loss of signal in the Alexa 488 channel after incubation in Exonuclease III for 1 hour at room temperature. The right image shows re-amplification of the Actb transcript after adding only the new bridging strand and the corresponding hairpin labeled with Alexa 647 dye. The contrast of the images was adjusted to illustrate certain features of the method.

[0467] In some embodiments, Lambda exonuclease (λ-exo) is used to remove detectably labeled oligonucleotides. Fig. 22 An exemplary method of HCR rehybridization using λ-exo is shown. Fig. 22 In the process, λ-exo digests the 5' phosphorylated bridging strand and releases the HCR polymer from the intermediate oligonucleotide bound to the target (eg, mRNA), and keeps the intermediate oligonucleotide intact for hybridization with a new bridging strand after washing away the released polymer. Fig. 22 (b) shows exemplary data using detectably labeled oligonucleotides targeting the β-actin (Actb) transcript in T3T mouse fibroblasts. The left image shows the initial hybridization and amplification of the Actb transcript using Alexa 488 dye. The middle image shows the loss of signal in the Alexa 488 channel after incubation in λ-exo for 1 hour at 37°C. The right image shows the re-amplification of the Actb transcript after washing with wash buffer and adding only the new bridging strand together with the corresponding hairpin labeled with Alexa 647 dye. The contrast of the images was adjusted to illustrate certain features of the method.

[0468] In some embodiments, a uracil-specific excision reagent (USER) is used to remove detectably labeled oligonucleotides. Fig.23 An exemplary method of HCR rehybridization using USER is shown. Fig.23In , USER digests at the deoxyuridine nucleotides in the bridging strand and releases the HCR polymer from the intermediate oligonucleotide bound to the target (e.g., mRNA), and keeps the intermediate nucleotide intact for hybridization with a new bridging strand after washing away the fragments and released polymer. Fig.23 (b) shows exemplary data using detectably labeled oligonucleotides targeting β-actin (ACTb) in T3T mouse fibroblasts. The left image shows initial hybridization and amplification of the Actb transcript using Alexa 488 dye. The middle image shows the loss of signal in the Alexa 488 channel after incubation in USER for 1 hour at 37°C. The right image shows the re-amplification of the Actb transcript after washing with wash buffer and adding only the new bridging strand together with the corresponding hairpin labeled with Alexa 647 dye. The contrast of the images was adjusted to illustrate certain features of the method.

[0469] In some embodiments, detectably labeled oligonucleotides are removed by replacement with complementary oligonucleotides (cTOE). In some embodiments, replacement includes the use of dextran or its derivatives, salts and / or organic solvents. In some embodiments, replacement includes the use of dextran or its derivatives. In some embodiments, replacement includes the use of dextran sulfate. In some embodiments, replacement includes the use of salt. In some embodiments, the salt is MgCl2. In some embodiments, replacement includes the use of organic solvents. In some embodiments, the organic solvent is formamide. Multiple factors can be optimized individually or in combination, such as but not limited to cTOE concentration, incubation time, composition of buffer and type and / or concentration of organic solvent. Fig.24 Shown are exemplary data of using cTOE to replace smFISH probes. Shown are the average ratios of fluorescence intensity between the smFISH probes to be replaced (Alexa 647) and the colocalized smFISH probes (Alexa 532). Performed multiple treatments, wherein the concentration of cTOE, hybridization buffer composition, and displacement time were compared. All displacement probe conditions resulted in significantly more displacement than controls, in which cells were placed in 10% DS and cTOE was not added. Not intended to be limited by theory, the applicant noted that, among other things, increasing the concentration of cTOE, increasing the amount of time for cTOE probe hybridization, adjusting the buffer to 10mM MgCl or 10% formamide all resulted in increased displacement. cTOE at 2.5 μM in 10% dextran sulfate (DS) for 2 hours resulted in a minimal residual but smaller increase than the baseline signal of Alexa 647smFISH signal, which was determined by hybridizing Alexa 594 (A594) instead of Alexa 647 and not adding cTOE.

[0470] Additional Examples of Oligonucleotide Preparation

[0471] By PCR amplification of a set of sequences ( Fig.25 ). The product is separated, for example, precipitated at least 10 minutes at -20°C with 5 volumes of precipitation buffer (30:1 EtOH:1M NaAc). The precipitation mixture is centrifuged for 10 minutes. The supernatant is discarded and the oligonucleotide mass is reconstructed with an appropriate unit of enzyme in a nickase buffer, based on about 10 units of enzyme digestion of about 1 μg DNA in 1 hour. After the incubation time is over, the sample is precipitated again and reconstructed in 2X loading buffer (96% formamide / 20mM EDTA) and water to prepare the final loading buffer (48% formamide / 10mM EDTA). The sample is heated to 95°C to completely denature the DNA. The denatured DNA is then loaded into a denaturing polyacrylamide gel (8M urea 10-12% acrylamide). The gel is run at 250V for 1 hour, or optimized as desired. After electrophoresis, the gel is stained with 1x sybr gold for 15 minutes and then visualized. The appropriate band is cut out, broken, and incubated in DI water for 2 hours. After incubation, the sample was precipitated again and then purified using a vacuum column. The column was eluted with 30 μL of RNase-free water to obtain the final product, as in Fig.26 As shown in .

[0472] In some embodiments, provided methods use restriction enzyme sites instead of nicking endonuclease sites. Fig.25 Similar to the amplification steps in , a group of sequences with BamHI sites flanking the 5'-end and AatII sites flanking the 3'-end were amplified by PCR. The PCR product was precipitated at -20°C with 5 volumes of precipitation buffer (30:1EtOH:1M NaOAc) for at least 10 minutes and separated, and then digested with BamHI and AatII. The product was purified again and subjected to exo III digestion. Removal of digested nucleic acid provided product oligonucleotides.

[0473] Equivalent

[0474] Having described some illustrative embodiments of the present invention, it should be apparent to those skilled in the art that the above is merely illustrative and non-restrictive, and is presented only by way of example. A large number of modifications and other illustrative embodiments are within the purview of those of ordinary skill in the art and are conceived to fall within the scope of the present invention. In particular, although many of the examples presented herein relate to specific combinations of method behaviors or system elements, it should be understood that these behaviors and these elements can be combined in other ways to accomplish the same goal. The actions, elements, and features discussed only in conjunction with one embodiment are not intended to exclude similar roles in other embodiments. In addition, for one or more method-plus-function limitations cited in the following claims, the method is not intended to be limited to the method disclosed herein for performing the described function, but is intended to cover in scope any method now known or later developed for performing the described function.

[0475] The use of ordinal terms such as "first", "second", "third", etc., in the claims to modify claim elements does not by itself imply any priority, superiority, or order of one claim over another or the temporal order in which the acts of a method are performed, but is merely used as a marker to distinguish one claim element having a certain name from another element having the same name (but using an ordinal term) to distinguish the claim elements. Similarly, the use of a), b), etc., or i), ii), etc. in the claims does not by itself imply any priority, superiority, or order of steps in the claims. Similarly, the use of these terms in the description does not by itself confer any required priority, superiority, or order.

[0476] It is believed that the above written description is sufficient to enable those skilled in the art to practice the present invention. The present invention is not limited by the scope of the embodiments provided, as the embodiments are intended as a single illustration of one aspect of the present invention and other functionally equivalent embodiments are within the scope of the present invention. In addition to those shown and described herein, various changes of the present invention will become apparent to those skilled in the art based on the foregoing description and fall within the scope of the appended claims. The advantages and objects of the present invention are not necessarily included by each embodiment of the present invention.

Claims

1. A method for non-diagnostic purposes comprising the following steps: (a) contacting a cell comprising a plurality of nucleic acids with a first plurality of detectably labeled oligonucleotides comprising a first set of detectably labeled oligonucleotides such that the composition comprises at least: (i) a first detectably labeled oligonucleotide that interacts with a first target nucleic acid in the cell via one or more intermediate oligonucleotides; and (ii) a second detectably labeled oligonucleotide that interacts with a second target nucleic acid in the cell via one or more intermediary oligonucleotides; wherein the first detectably labeled oligonucleotide is different from the second detectably labeled oligonucleotide; (b) imaging the cell after the first contacting step to detect the interaction of the detectably labeled oligonucleotide with its target nucleic acid; (c) performing a second contacting step, the second contacting step comprising contacting the cell with a second plurality of detectably labeled oligonucleotides, the second plurality of detectably labeled oligonucleotides comprising: (i) a third detectably labeled oligonucleotide that interacts with the first target nucleic acid through one or more intermediate oligonucleotides; and (ii) a fourth detectably labeled oligonucleotide that interacts with the second target nucleic acid via one or more intermediary oligonucleotides, wherein the third detectably labeled oligonucleotide is labeled with a detectable moiety different from that of the first detectably labeled oligonucleotide, or the fourth detectably labeled oligonucleotide is labeled with a detectable moiety different from that of the second detectably labeled oligonucleotide; or the third detectably labeled oligonucleotide is labeled with a detectable moiety different from that of the first detectably labeled oligonucleotide and the fourth detectably labeled oligonucleotide is labeled with a detectable moiety different from that of the second detectably labeled oligonucleotide; (d) imaging the cells after the second contacting step to detect the interaction of the detectably labeled oligonucleotides with their targets; and (e) repeating the contacting and imaging steps, each time using a new plurality of detectably labeled oligonucleotides comprising detectably labeled oligonucleotides that target overlapping nucleic acids targeted by the first and second plurality of detectably labeled oligonucleotides through one or more intermediate oligonucleotides, wherein each plurality of detectably labeled oligonucleotides utilized differs from each other plurality of detectably labeled oligonucleotides utilized by at least one difference in the detectable portion of the nucleotides that are labeled to target the same transcript; The detected signal is caused to barcode the first target nucleic acid, and the second target nucleic acid, wherein the barcodes are distinguishable from each other.

2. The method of claim 1, wherein at least one contacting step differs from another contacting step in terms of the labeling of at least one of the first target nucleic acid and the second target nucleic acid.

3. The method of claim 1, wherein each detectably labeled oligonucleotide comprises a detectable moiety and at least one contacting step differs from another contacting step in having a different detectable moiety for the first target nucleic acid or for the second target nucleic acid.

4. The method of claim 1, wherein at least two different detectably labeled oligonucleotides interact with a first target nucleic acid, and wherein at least two different detectably labeled oligonucleotides interact with a second target nucleic acid.

5. The method of claim 2, wherein at least two contacting steps differ from each other with respect to the labeling of the first target nucleic acid, and at least two contacting steps differ from each other with respect to the labeling of the second target nucleic acid.

6. The method of claim 5, wherein at least five different detectably labeled oligonucleotides interact with the first target nucleic acid, and wherein at least five different detectably labeled oligonucleotides interact with the second target nucleic acid.

7. The method of claim 2, wherein the target nucleic acid is a cellular RNA or a DNA locus.

8. The method of claim 2, wherein the target nucleic acid is a transcript.

9. The method of claim 2, wherein each of the detectably labeled oligonucleotides comprises the same label.

10. The method of claim 1, wherein the detectably labeled oligonucleotides have labels that differ from one another.

11. The method of claim 2, wherein the detectably labeled oligonucleotide comprises a label selected from 2, 3, or 4 different labels.

12. The method of claim 1, wherein the intermediate oligonucleotide comprises a sequence complementary to its target nucleic acid, an overhang sequence, or a combination thereof.

13. The method of claim 12, wherein the overhang sequence is complementary to a detectably labeled oligonucleotide.

14. The method of claim 12, wherein the overhang sequence is complementary to a bridging oligonucleotide.

15. The method of claim 14, wherein the bridging oligonucleotide is complementary to a detectably labeled oligonucleotide.

16. The method of claim 1, wherein the intermediate oligonucleotide is retained throughout the multiple contacting and imaging steps.

17. The method of claim 1 or 2, wherein each repeated imaging step comprises imaging the cell after the repeated contacting step to detect hybridization by the plurality of detectably labeled oligonucleotides.

18. The method of claim 1 or 2, wherein the detectably labeled oligonucleotide is removed after one or more imaging steps.

19. The method of claim 18, wherein the step of removing comprises contacting the plurality of detectably labeled oligonucleotides with an enzyme that digests detectably labeled oligonucleotides.

20. The method of claim 18, wherein the step of removing comprises contacting the plurality of detectably labeled oligonucleotides with a DNase, photobleaching, strand displacement, formamide washing, heat denaturation, or a combination thereof.

21. The method of claim 18, wherein the detectably labeled oligonucleotide is removed by photobleaching.

22. The method of claim 1, wherein the barcode of the target nucleic acid in the cell is different in the absence of the target nucleic acid signal in the cell.

23. The method of claim 1 or 2, wherein the at least one contacting step differs from another contacting step in labeling at least one of the first target nucleic acid and the second target nucleic acid in that there is no signal for at least one of the first target nucleic acid and the second target nucleic acid.

24. The method of claim 2, wherein the at least one contacting step differs from another contacting step in at least one aspect of labeling the first target nucleic acid and the second target nucleic acid by having different detectably labeled oligonucleotides for the first target nucleic acid and the second target nucleic acid.

25. The method of claim 2, wherein the at least one contacting step differs from another contacting step in at least one aspect of labeling the first and second target nucleic acids by having different intermediate oligonucleotides for the first and second target nucleic acids.

26. The method of claim 2, wherein the at least one contacting step differs from another contacting step in at least one aspect of labeling the first target nucleic acid and the second target nucleic acid by having different labels for the first target nucleic acid and the second target nucleic acid.

27. The method of claim 1 or 2, wherein the barcode of the target nucleic acid in the cell does not include a signal for one or more contacting steps.

28. The method of claim 1 or 2, wherein the signal is amplified.

29. The method of claim 1 or 2, wherein the signal is amplified by hybridization chain reaction.

30. The method of claim 1 or 2, wherein the method targets up to F N target nucleic acids, where F is the number of types of detectable moieties and N is the number of detectably labeled oligonucleotides of the target.

31. The method of claim 30, wherein the method comprises N contacting steps.

32. The method of claim 30, wherein the method comprises more than N contacting steps.

33. The method of claim 30, wherein the barcode of the target nucleic acid does not include a signal from one or more contacting steps.

34. The method of claim 2, wherein the cells are cleared.

35. The method of claim 34, wherein the cells are clarified by CLARITY, CLAMPFISH, bDNA, or hydrogel.

36. The method of claim 1 or 2, wherein each detectably labeled oligonucleotide comprises two or more detectable moieties.

37. The method of claim 1, wherein the first target nucleic acid is different from the second target nucleic acid.

38. The method of claim 1, wherein the first target nucleic acid is different from the second target nucleic acid.

39. The method of claim 23, wherein the first target nucleic acid is different from the second target nucleic acid.

40. The method of claim 1, wherein each of the detectably labeled oligonucleotides comprises the same label.

41. The method of claim 1 or 2, wherein each of the detectably labeled oligonucleotides comprises the same label and the same sequence.

42. The method of claim 1, wherein each detectably labeled oligonucleotide interacts with its target nucleic acid through one or more intermediary oligonucleotides, each of the one or more intermediary oligonucleotides hybridizing to the target nucleic acid.

43. The method of claim 37, wherein the at least one contacting step differs from another contacting step in labeling at least one of the first and second nucleic acid targets by having no signal for at least one of the first and second nucleic acid targets.

44. The method of claim 37, wherein at least one of the contacting steps differs from another contacting step in labeling at least one of the first target nucleic acid and the second target nucleic acid by having different detectably labeled oligonucleotides for the first target nucleic acid or the second target nucleic acid.

45. The method of claim 37, wherein at least one of the contacting steps differs from another contacting step in labeling at least one of the first and second target nucleic acids by having a different intermediary oligonucleotide for the first or second target nucleic acid.

46. ​​The method of claim 37, wherein at least one of the contacting steps differs from another contacting step in terms of labeling at least one of the first and second target nucleic acids by having a different label for the first or second target nucleic acid.

47. The method of claim 1, wherein one new set of multiple detectably labeled oligonucleotides comprises a new detectably labeled oligonucleotide that interacts with the first target nucleic acid that is different from the first detectably labeled oligonucleotide, and one new set of multiple detectably labeled oligonucleotides comprises another new detectably labeled oligonucleotide that interacts with the second target nucleic acid that is different from the second detectably labeled oligonucleotide.

48. The method of claim 1, wherein the cell comprises a tissue.

Images