Targeted Expansion Fluorescent In Situ Sequencing
RELATED APPLICATION DATA
 This application claims priority to U.S. Provisional Application No. 62/516,327 filed on June 7, 2017, which is hereby incorporated by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENT INTERESTS
 This invention was made with government support under D16PC0008 awarded by the Intelligence Advanced Research Projects Activity (IARPA). The U.S. government has certain rights in the invention.
TECHNICAL FIELD OF THE INVENTION
 This invention is related to the areas of histology, immunohistochemistry, hydrogel chemistry, is situ hybridization, tissue clearing, expansion microscopy, cryosectioning, and fluorescent in situ sequencing.
BACKGROUND OF THE INVENTION
 Fluorescent In Situ Sequencing (FISSEQ) is a powerful technique that allows the sequence of a nucleic acid to be determined while maintaining its location within a biological sample. FISSEQ has been performed in a variety of cell and tissue types and can simultaneously probe many sequences in parallel. FISSEQ workflow involves sample fixation followed by the amplification of the nucleic acid(s) of interest. Rolling circle amplification is commonly used to create a dense nanoball of DNA, called a rolony. Next-generation sequencing is then performed on the rolonies. In instances where the rolony density is high, resolving the identity and location can be difficult and new methods are needed to de-crowd the signals.
 Expansion Microscopy (ExM) is a technique which embeds a biological sample into a swellable polymer matrix which can physically enlarge the specimen. Physical
expansion creates a virtual increase in magnification and allows super resolution imaging to be accomplished on diffraction -limited microscopes. By combining FISSEQ with ExM, one can obtain the location and sequence of nucleic acids of interest at high resolution (ExSEQ).
 There is a continuing need in the art to improve techniques for analyzing nucleic acids.
SUMMARY OF THE INVENTION
 According to one aspect of the invention a method of preparing a tissue sample for microscopy is provided. A tissue sample comprising RNA is contacted with a reverse transcriptase enzyme and an activated DNA primer that is complementary to one or more specific targets in the RNA. The tissue sample is incubated under conditions suitable for reverse transcription, forming activated cDNA molecules. A first gelling solution is added to the tissue sample; the first gelling solution comprises reagents necessary for forming an expandable cross-linked polymer. The tissue sample is incubated under conditions suitable for polymerization of the cross-linked polymer. An expandable cross-linked polymer is formed that comprises the cDNA molecules in covalent linkage.
 According to another aspect of the invention a method of preparing a tissue sample for microscopy is provided. A brain tissue sample comprising RNA is contacted with a reverse transcriptase enzyme and an activated locked nucleic acid primer that is complementary to one or more specific targets in the RNA. The tissue sample is incubated under conditions suitable for reverse transcription, forming activated cDNA molecules. A first gelling solution is added to the brain tissue sample. The first gelling solution comprises reagents necessary for forming an expandable cross-linked polymer. The brain tissue sample is incubated under conditions suitable for polymerization of the cross-linked polymer. An expandable cross-linked polymer is formed that comprises the cDNA molecules in covalent linkage. The expandable cross-linked polymer is expanded by adding water to it, forming an expanded cross- linked polymer. A padlock probe is added to the tissue sample. The cDNA molecules are then subjected to rolling circle amplification.
 Another aspect of the invention is a composition comprising a tissue sample and a nucleic acid primer. The tissue sample is embedded in an expandable cross-linked polymer. The tissue sample comprises RNA. The nucleic acid primer is complementary to one or more specific targets in the RNA. The nucleic acid primer is covalently linked to the expandable cross-linked polymer.
 Yet another aspect of the invention is a composition comprising a tissue sample and a nucleic acid primer. The tissue sample is embedded in an expanded, cross-linked polymer. The tissue sample comprises RNA. The nucleic acid primer is complementary to one or more specific targets in the RNA. The nucleic acid primer is covalently linked to the expanded cross-linked polymer.
 Still another aspect of the invention is a composition comprising a tissue sample and cDNA. The cDNA is reverse transcribed from a specific subset of transcripts in the tissue sample. The tissue sample is embedded in an expandable cross-linked polymer. The cDNA is covalently linked to the expandable cross-linked polymer.
 A further aspect of the invention is a composition comprising a tissue sample and cDNA. The cDNA is reverse transcribed from a specific subset of transcripts in the tissue sample. The tissue sample is embedded in an expanded cross-linked polymer. The cDNA is covalently linked to the expanded cross-linked polymer.
 These and other aspects and embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with sensitive tools for analyzing genomes and transcriptomes.
BRIEF DESCRIPTION OF THE DRAWINGS
 Fig. 1. Low magnification (lOx) image of Sindbis injected brain slice used for ExSEQ.
 Fig. 2. Low magnification (10x) images of three rounds of in situ SOLID sequencing in ~3x expanded brain tissue. Inserts show the change in color per iteration.
 Fig. 3A-3B. Confocal z stack images of ExSEQ. The Fig. 3 A image captures cells of all four colors while only green and red cells are visible in the Fig. 3B image.
 Fig. 4A-4B. Maximum intensity projections from images in Fig. 3. The Fig. 4A image captures cells of all four colors while only green and red cells are visible in the Fig. 4B image. Inserts show areas where cell projections are in close proximity with each other.
DETAILED DESCRD7TION OF THE INVENTION
 The inventors have developed an Expansion Sequencing (ExSEQ) method which is useful for interrogating one or more specific targets of interest. The targeted ExSEQ method typically utilizes a padlock probe in Fluorescent In Situ Sequencing (FISSEQ). In this method, RNA (including but not limited to mRNA, microRNA, or long noncoding RNA) may be reverse transcribed to produce cDNA. A DNA probe (padlock probe) may then be added which can cyclize onto the region of interest on the cDNA. Ligation of this probe followed by rolling circle amplification produces amplified cDNA which can be sequenced.
 To incorporate a Fluorescent In Situ Sequencing workflow into an expandable polymer network for analysis in Expansion Microscopy (ExM), a reactive handle is introduced into the sample. One can introduce this handle onto the 5' end of a reverse transcription nucleic acid primer (including but not limited to a primer containing a locked nucleic acid (LNA) ribonucleotide. After reverse transcription, the sample may be infused with a polymerizable solution and gelled. The sample may be digested, expanded, mounted onto a slide, and chemically pacified to render the polymer matrix inert. Rolling circle amplication may be applied to the cDNA, producing "rolonies" that may be sequenced at high resolution. The order of steps may vary, as may the actual types of nucleic acid polymerization, transcription, and gel polymerization used.
 Activated primers for use in reverse transcription are functionalized so that they can be incorporated into a polymer matrix. Chemical modifications which are suitable for activating primers include, but are not limited to, polymerizable handles, electrophile
handles, nucleophile handles, and other reactive handles. Polymerizable handles include, but are not limited to, methacrylate, acrylate, acrylamide, methacrylamide, radical initiating, controlled radical polymerization handles, and vinyl sulphone groups. Electrophile handles include, but are not limited to, aldehydes, ketones, maleimides, thioesters, alpha-iodo carbonyls, vinyl sulphone groups, and carboxylates. Nucleophile handles include, but are not limited to, amino and thiol groups. Other reactive handles include, but are not limited to, azido, alkynyl, strained alkene, strained alkyne, and tetrazine groups. These reactive groups permit the incorporation of the extended reverse transcription primers into expandable cross- linked polymer matrices. The incorporation may conveniently be done upon formation of the matrix. Alternatively, it can be incorporated before or after formation of the matrix, for example, by reaction with a monomer or oligomer reactant. When reverse transcription occurs, the chemical modification that was on the primer ends up on the product cDNA, because the primer is extended to form the cDNA.
 The primer will be complementary to a specific gene, genes, transcript, or transcripts.
The primer has a region of complementarity to one or more targets (genes or transcripts). The primer region may be complementary to between 1 and 100 targets inclusive, between 1 and 75 targets, between 1 and 50 targets, between 1 and 25 targets, between 1 and 20 targets, between 1 and 15 targets, between 1 and 10 targets, between 1 and 5 targets. The primer may be complementary to one target. The specific gene, genes, transcript, or transcripts are a specific subset of the genes or transcripts in the tissue sample. The entire genome or transcriptome is not transcribed or reverse transcribed to make a specific subset. Thus the primers that are used are not ones that are random in sequence or bind to all or essentially all genes or transcripts.
 A gelling solution for creating an expandable polymer matrix may be formed using any known chemistry. The expandable polymer matrix will typically be transparent, so that it does not impair observation by microscopy. The expandable polymer matrix will typically expand isotropically, so that the same 3 -dimensional relationships are maintained as before expansion. Monomers or oligomers for forming a polymer
matrix may be substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinyl alcoholos, vinalmines, allylamines, allylalcohols, etc. Polymer matrices may be formed by cross-linking of oligomers or polymers.
 Tissue samples may be obtained from any source. These may be from animal, plant, or bacteria. The tissue may derive from any organ or cell source in an organism. Exemplary tissues are brain, neuronal, spinal, peripheral nerve, muscle, bone marrow, heart, lung, breast, prostate, pancreas, colorectal, stomach, gall bladder, retina, skin, esophagus, ovary, uterus, testes, and fallopian tube. Tissues may be from healthy or diseased organisms or organs.
 Digestion of structural components of the tissue samples may be accomplished using any enzymatic, chemical, or mechanical means known in the art. Proteases may be used to digest proteins. Destruction or loosening of the structural components facilitates the swelling of the tissues upon swelling of the expandable polymer matrices.
 The second gelling solution is added to the tissue samples after the swelling of the first gelling solution. Alternatively, the second gelling solution is present with the first gelling solution but is not activated to form a gel until after the swelling of the first gel. The second gel is used to provide protective solidity to the tissue sample after expansion.
 Primers that comprise locked nucleic acids are those that comprise a ribose moiety modified with an extra bridge connecting the 2' oxygen and 4' carbon. This conformational restraint increases the binding affinity of complementary nucleic acids.
 Padlock probes contain two regions of complementarity, one at each of their ends, to two adjacent regions of a target nucleic acid. When the padlock probe hybridizes to the target, it circularizes with its two ends adjacent, but not joined. A ligation reaction closes the opening between the two ends forming a closed circle. The closed circle can be used as a template for rolling circle amplification. Rolling circle amplification
may be performed as is known in the art. Typically this is accomplished with a DNA polymerase, such as Phi29 DNA polymerase.
 Nanoballs of DNA that are formed in situ in the expanded tissues in the gel matrices may be subjected to any type of nucleic analysis known in the art. A massively parallel sequencing technique can be used. Preferably the technique will yield fluorescent products that can be analyzed microscopically. The in situ analysis of nucleic acids can provide information on single cells, their genome and/or transcriptome.
 Swelling of polymer matrix may be accomplished by addition of water or other solvent. The polymer matrix absorbs the liquid and swells in an equivalent manner in each of three dimensions. The degree of swelling may be to a size that is at least two times, at least three times, at least four times, at least five times, or at least ten times the original, in each dimension. A non-swellable polymer matrix can be converted to a swellable polymer matrix by chemical treatment. This treatment would change a neutral matrix to a charged matrix and thus make it swellable. This chemical treatment can be performed at any step post-polymerization.
 The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
 Protocol for tissues transferred to charged glass slides:
 Most steps are li sted as washes with reagents for a designated period of time
PBST = PBS + 0.5% Tween-20
 DAY 1
pepsin 2mg/ml (0.2%) in 0.1M HC1 at room temp for 3 min for 14um slices. PBSTx2
70% ethanol 5min
85% ethanol 5min
100% ethanol 5min
100% ethanol lhr at 4C
Add RT soln and incubate at 37C overnight
RT soln for 200ul
2ul acrylated LNA RT primer (100 uM)
20ul RevertAid H Minus M-MuLV RT (200 U/ul)
4ul dNTPs (25 mM)
2ul BSA (20 ug/ul)
5ul RiboLock RNase Inhibitor (40U/ul)
20ul RT buffer (lOx)
147ul H20  DAY 2
40ul BS(PEG)9 inl60ul PBST at for 1 hour at room temperature
lMTris-HCl 8.0 wash
1M Tris-HCl 8.0 for 30min at room temperature PBSTx2
Remove flow chamber
Trim tissue with a razor to isolate only the region of interest
Place scotch tape on the sides of the slide flanking the tissue
Put gelling solution onto the sides of the slice and place a cover slip on top so that the tissue is in between the slide and coverslip
Add Gelling solution and incubate for 90min at 37C Gelling solution: for 200 ul 188ul Monomer solution
4ul 4-hydroxy-TEMPO (0.5%)
4ul TEMED (10%)
4ul ammonium persulfate (10%)
Monomer solution: for 9.4 mL
2.25mL Sodium acrylate (380 mg/mL)
0.5 mL Acrylamide (500 mg/mL)
0.75 mL N,N'-Methylenebisacrylamide (20 mg/mL)
4 mL Sodium Chloride (292 mg/mL)
1 mL PBS (lOx)
0.9 mL Water
Remove coverslip and trim excess gel with a razor blade Add gel to Digestion solution and incubate at 37C overnight
Digestion solution: for 3 mL
50 mM Tris pH 8.0
1 mM EDTA,
0.5% Triton X-100,
0.8 M guanidine HCl
Proteinase K (8 units/mL)
 DAY 3
Transfer gel to bind-silane treated slide Add water until gel is fully expanded
Add Re-embedding solution, place coverslip in gel, and incubate at 37C for 90 min
Re-embedding solution: for 1.8 mL
150 uL 19:1 Acrylamide: N,N-Methylenebisacrylamide (40%)
10 uL Tris base (1M)
15 uL TEMED (10%)
15 uL APS (10%)
1610 uL Water
Trim gel with razor to desired size
Place silicone gasket around gel
Add Passivation solution 1 and incubate at room temperature for 2 hours Passivation solution 1 : for 200 uL 100 uL Ethanolamine HC1 (4M) 100 uL MES buffer pH 6.5 (200 mM)
EDC 6 mg NHS 3 mg
Add Passivation solution 2 and incubate at room temperature for 40 min Passivation solution 2: for 200 uL 100 uL Ethanolamine HC1 (4M)
100 uL Sodium borate buffer pH 8.5 (125 mM)
Add Padlock solution and incubate for 30 min at 37C and then for 45 min at 45C
Padlock solution: for 200ul
20ul Ampligase buffer (lOx)
0.2ul /5p/padlock (100 uM) lul Ampligase (lOOU/ul)
0.4ul dNTP (25mM)
16ul RNase H (5U/ul)
20ul Phusion DNA polymerase (2U/ul)
5ul RiboLock RNase Inhibitor (40U/ul)
20ul KC1 stock solution (0.5M)
109.4ul H20 PBSTx2
Add RCA solution and incubate overnight at room temperature RCA solution: for 200ul
20ul phi29 polymerase (1 OU/ul)
20ul phi29 polymerase buffer (lOx)
2ul dNTPs (25 mM)
2ul BSA (20 ug/ul)
20ul glycerol (50%) lul aadUTP (4mM)
 DAY 4
40ul BS(PEG)9 inl60ul PBST at RT for lhr lMTris-HCl 8.0 wash
1M Tris-HCl 8.0 for 30min at room temperature 2xSSC, 10% formamide x3
For rolony detection - add hybridization probe at 2.5 uM in 2xSSC, 10% formamide
For sequencing - add primer at 2.5 uM in 2xSSC, 10% formamide, wash, and proceed to sequencing
 Preliminary Results
 The protocol was validated on murine brain slices which contain neurons infected by a sindbis virus. Sindbis Infected neurons express GFP (Figure 1) and contain many copies of an RNA with a randomized sequence flanked by a constant sequence. The virus library is prepared such that each cell contains multiple copies of only one unique sequence. The ExSEQ procedure was performed on this tissue using an LNA primer for the constant sequence. Library preparation was performed followed by three iterations of SOLID sequencing by ligation. Throughout these three iterations it was observed that the rolonies within each neuron were the same color per iteration while rolonies in different neurons changed color (Figure 2). This result is consistent with each cell having multiple copies of a unique RNA sequence. For the first iteration of SOLID™ sequencing, only two colors were visible because the first two bases in the barcode are pyrimidine-pyrimidine. Higher magnification imaging was performed on a confocal microscope to get a clearer idea of rolony density and location. Figure 3 shows stills of three dimensional images showing that the rolony density is high enough to capture cell morphology. Maximum intensity projections (Figure 4) of the z stacks additionally provide enough resolution to distinguish rolonies of adjacent cells.
The disclosure of each reference cited is expressly incorporated herein. Boyden et al., US 2016/0305856 Al
Lee et al., "Highly multiplexed subcellular RNA sequencing in situ, " Science, 2014, 343:1360-1363
Boyden et al., US 2016/0304952
Chen et al., US 2016/0116384
Wassie et al., US 201/0067096