Abstract: Among other things, this disclosure provides a method of detecting a target nucleic acid. Aspects of the method include: (a) obtaining a labeled nucleic acid probe that is complementary to a target nucleic acid, wherein the probe comprises a capture tag; (b) hybridizing the probe with the target nucleic in a fixed cell, in situ, to produce a duplex; (c) linking the probe in the duplex to a peroxidase conjugate via the capture tag to produce a peroxidase-labeled duplex; and (d) incubating the peroxidase-labeled duplex with a peroxidase substrate, wherein the peroxidase activity of the peroxidase conjugate catalyzes deposition of the substrate in the vicinity of the duplex, thereby producing a detectable signal.

Abstract: A method of processing an RNA sample is provided. In certain embodiments, the method may comprise: a) obtaining a fragmented RNA sample comprising: i. RNA fragments of long RNA molecules; and ii. unfragmented short RNA; and b) contacting said fragmented RNA sample with a first adaptor in the presence of a RtcB ligase, thereby producing a ligated RNA sample comprising adaptor-ligated fragments of long RNA. A kit for performing the method is also provided.

Abstract: This disclosure provides, among other things, a method for analyzing a planar cellular sample. In some embodiments, the method comprises: (a) indirectly or directly attaching nucleic acid tags to binding sites in a planar cellular sample; (b) contacting the planar cellular sample with a solid support comprising an array of spatially addressed features that comprise oligonucleotides, wherein each oligonucleotide comprises a molecular barcode that identifies the feature in which the oligonucleotides is present; (c) hybridizing the nucleic acid tags, or a copy of the same, with the oligonucleotides to produce duplexes; and (d) extending the oligonucleotides in the duplexes to produce extension products that each comprises (i) a molecular barcode and (ii) a copy of a nucleic acid tag. Other embodiments, e.g., kits and the like, are also described.

Abstract: Provided herein is a method of sample analysis. In certain embodiments, the method comprises: a) cross-linking protein of a cell using a first compound to produce a first cross-linked product comprising cross-linked protein, and RNA; b) contacting the first cross-linked product and a second compound under conditions by which an oligonucleotide portion of the second compound hybridizes to the RNA; c) activating a reaction the first and second compound, thereby covalently crosslinking the oligonucleotide to the cross-linked protein to produce a second cross-linked product; d) isolating the second cross-linked product using an affinity tag; and e) analyzing the isolated second cross-linked product. Compounds for performing the method are also provided.

Abstract: Provided herein is a method of sample analysis. In certain embodiments, the method comprises: a) cross-linking protein of a cell using a first compound to produce a first cross-linked product comprising cross-linked protein, and RNA; b) contacting the first cross-linked product and a second compound under conditions by which an oligonucleotide portion of the second compound hybridizes to the RNA; c) activating a reaction the first and second compound, thereby covalently crosslinking the oligonucleotide to the cross-linked protein to produce a second cross-linked product; d) isolating the second cross-linked product using an affinity tag; and e) analyzing the isolated second cross-linked product. Compounds for performing the method are also provided.

Abstract: Provided herein is a method of preparing an RNA sample comprising: a) obtaining an RNA sample comprising: i. long RNA molecules that may be unfragmented or fragmented to contain 5?-OH group and a 2?-3?-cyclic phosphate group; and ii. short RNA molecules that comprise a 5? phosphate group and a 3? OH group; and b) contacting the RNA sample with an adaptor comprising either a 2?-PO group and 3?-OH group or a 2?,3?-cyclic phosphate group in the presence of a eukaryotic tRNA ligase, thereby producing a ligated RNA sample in which a) the short RNA molecules are selectively ligated to the adaptor or b) the short RNA molecules and long RNA fragments are selectively ligated to the adaptor.

Abstract: A method of enriching for a fragment of a genome, as well as corresponding compositions and kits, are provided. In certain embodiments, the method comprises: (a) contacting a sample comprising fragmented DNA with a Cas9-gRNA complex comprising mutant Cas9 protein that has inactivated nuclease activity and a Cas9-associated guide RNA that is complementary to a site in the DNA, to produce a Cas9-fragment complex that comprises a fragment of the fragmented DNA; and (b) isolating the complex. In addition, other methods and compositions for Cas9/CRISPR-mediated nucleic acid manipulation are also provided.

Abstract: A method for fragmenting a genome is provided. In certain embodiments, the method comprises: (a) combining a genomic sample containing genomic DNA with a plurality of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes comprise a Cas9 protein and a set of at least 10 Cas9-associated guide RNAs that are complementary to different, pre-defined, sites in a genome, to produce a reaction mixture; and (b) incubating the reaction mixture to produce at least 5 fragments of the genomic DNA. Also provided is a composition comprising at least 100 Cas9-associated guide RNAs that are each complementary to a different, pre-defined, site in a genome. Kits for performing the method are also provided. In addition, other methods, compositions and kits for manipulating nucleic acids are also provided.

Abstract: Provided herein is method comprising: contacting an initial RNA sample containing a population of different RNA molecules with a divalent cation and a set of DNAzymes that are designed to cleave multiple target RNAs in the initial sample, thereby producing a product RNA sample that comprises: a) uncleaved RNA molecules and b) cleaved RNA fragments that contain a 2?,3?-cyclic-phosphate and a 5? hydroxyl as the result of DNAzyme cleavage.

Abstract: Provided herein is a proximity assay that, in certain embodiments, involves: (a) hybridizing a first oligonucleotide and a second oligonucleotide with a target nucleic acid, wherein the first oligonucleotide comprises: i. a region that is complementary to a first sequence in the target nucleic acid and ii. a barcode sequence; and the second oligonucleotide comprises i. a region that is complementary to a second region in the target and ii. the complement of the barcode sequence; and (b) detecting hybridization between the barcode sequence and the complement of the barcode sequence, wherein hybridization between the barcode sequence and the complement of the barcode sequence indicates that the first and second target sequences are proximal to one another in the sample.

Abstract: This disclosure provides a method comprising: a) clamping the top and bottom strands of a double stranded DNA molecule to produce a duplex in which the top and bottom strands are linked; b) denaturing the duplex to produce a denatured product; and c) renaturing the denatured product in the presence of a labeled oligonucleotide that is complementary to a sequence of nucleotides in the double stranded DNA molecule, thereby producing a D-loop-containing product. Kits for performing the method and products made by the method are also provided.

Abstract: A method of processing an RNA sample is provided. In certain embodiments, the method may comprise: a) obtaining a fragmented RNA sample comprising: i. RNA fragments of long RNA molecules; and ii. unfragmented short RNA; and b) contacting said fragmented RNA sample with a first adaptor in the presence of a RtcB ligase, thereby producing a ligated RNA sample comprising adaptor-ligated fragments of long RNA. A kit for performing the method is also provided.

Abstract: Provided herein is a method of preparing an RNA sample comprising: a) obtaining an RNA sample comprising: i. long RNA molecules that may be unfragmented or fragmented to contain 5?-OH group and a 2?-3?-cyclic phosphate group; and ii. short RNA molecules that comprise a 5? phosphate group and a 3? OH group; and b) contacting the RNA sample with an adaptor comprising either a 2?-PO group and 3?-OH group or a 2?,3?-cyclic phosphate group in the presence of a eukaryotic tRNA ligase, thereby producing a ligated RNA sample in which a) the short RNA molecules are selectively ligated to the adaptor or b) the short RNA molecules and long RNA fragments are selectively ligated to the adaptor.

Abstract: Provided herein is a method of sample analysis. In certain embodiments, the method comprises: a) cross-linking protein of a cell using a first compound to produce a first cross-linked product comprising cross-linked protein, and RNA; b) contacting the first cross-linked product and a second compound under conditions by which an oligonucleotide portion of the second compound hybridizes to the RNA; c) activating a reaction the first and second compound, thereby covalently crosslinking the oligonucleotide to the cross-linked protein to produce a second cross-linked product; d) isolating the second cross-linked product using an affinity tag; and e) analyzing the isolated second cross-linked product. Compounds for performing the method are also provided.

Abstract: A method for partitioning a genome is provided. In certain embodiments, the method comprises: a) nicking a region of the genome using a sequence-specific nicking endonuclease to produce a nicked double-stranded genomic region; b) hybridizing the nicked double-stranded genomic region with an oligonucleotide comprising: i. an affinity tag; and ii. a nucleotide sequence that is complementary to the nucleotide sequence that is immediately adjacent to the nick site, to produce a duplex in which a terminal nucleotide of the oligonucleotide lies immediately adjacent to said a nucleotide of the nick site; c) ligating the terminal nucleotide of the oligonucleotide to the nucleotide of the nick site to produce a ligation product; and d) separating the ligation product from unligated products using the affinity tag. Compositions and kits for practicing the method are provided.

Abstract: A method of sample analysis is provided. The method comprises: a) contacting a genomic sample comprising a plurality of intact chromosomes, i.e., metaphase or interphase chromosomes, with a first set of labeled oligonucleotide probes under in situ hybridization conditions to produce a contacted sample comprising labeled chromosomes, where i. each of the labeled oligonucleotide probes is complementary to a non-repetitive, unique sequence in a region that flanks the centromere of a single chromosome of the plurality of chromosomes; and ii. hybridization of the labeled oligonucleotide probes to the chromosomes produces a distinct labeling pattern for each hybridized chromosome, thereby allowing each of the labeled chromosomes to be distinguished from one another; b) imaging the hybridized chromosomes to provide an image showing the labeling pattern for each labeled chromosome; and c) enumerating a labeled chromosome based on the labeling pattern of said labeled chromosome.

Abstract: A method of genome analysis is provided. In certain embodiments, the method may comprise: labeling the test genome using a first site-specific methyltransferase to produce a labeled test genome comprising a label; and analyzing the labeled test genome to determine if the test genome comprises a sequence alteration relative to a reference sequence. In certain embodiments, the method may comprise: evaluating binding of the labeled test genome to an array of probes, or observing a pattern of labeling along the labeled test genome.

Abstract: A method for partitioning a genome is provided. In certain embodiments, the method comprises: a) nicking a region of the genome using a sequence-specific nicking endonuclease to produce a nicked double-stranded genomic region; b) hybridizing the nicked double-stranded genomic region with an oligonucleotide comprising: i. an affinity tag; and ii. a nucleotide sequence that is complementary to the nucleotide sequence that is immediately adjacent to the nick site, to produce a duplex in which a terminal nucleotide of the oligonucleotide lies immediately adjacent to said a nucleotide of the nick site; c) ligating the terminal nucleotide of the oligonucleotide to the nucleotide of the nick site to produce a ligation product; and d) separating the ligation product from unligated products using the affinity tag. Compositions and kits for practicing the method are provided.

Abstract: A method of sample analysis is provided. In certain embodiments, the method may comprise: a) contacting a sample comprising a microbe with a set of at least two labeled oligonucleotides under in situ hybridization conditions to produce a contacted sample, where the labeled oligonucleotides i. hybridize to different RNA molecules of the microbe at sites that are unique to the microbe, ii. provide a predetermined optically detectable signature that identifies the microbe when the labeled oligonucleotides are hybridized to the different RNA molecules of the microbe, and iii. do not hybridize to ribosomal RNA of the microbe; b) reading the contacted sample to detect hybridization of the labeled oligonucleotides; and c) determining the identity of the microbe on the basis of the predetermined optically detectable signal, where the predetermined optically detectable signal indicates the identity of the microbe in the sample.

Abstract: A method of sample analysis is provided. In certain embodiments, the method may comprise: a) contacting under primer extension conditions a genomic sample comprising a test genome with a set of at least ten sequence-specific primers that are complementary to sites in only one strand of a reference chromosomal region, to produce primer extension products, and b) analyzing the primer extension products to identify a chromosomal rearrangement in the test genome.