Methods of analysis of a sample using hybridization chain reaction (HCR) are provided herein. Some embodiments involve one, two, or all three of the following aspects: 1) repeated signal detection, 2) overlapping binding sites, and 3) catalytic reporter deposition (CARD). Compositions and kits relating to these are also provided. Some embodiments encompass a method for repeated signal detection with reporter-labeled HCR hairpins involving providing a sample possibly containing one or more targets as well as possibly other molecules that are not targets, providing one or more probe sets each comprising either: a) one or more HCR initiator-labeled probes, or b) one or more probe units each comprising two or more HCR fractional initiator probes, providing one or more HCR amplifiers (each labeled with one or more reporters), detecting one or more signals from one or more reporters. In some embodiments, a probe unit comprises two or more HCR fractional initiator probes, wherein an HCR fractional initiator probe comprises a target-binding region and a fractional initiator, wherein the target-binding regions within a probe unit are configured to bind to overlapping or non-overlapping binding sites on the target, and wherein the fractional initiators on the probes within each probe unit are configured to bind to overlapping or non-overlapping binding sites on an HCR hairpin. Some embodiments encompass a method for HCR-mediated catalytic reporter deposition (CARD) for signal detection with hapten-labeled HCR hairpins involving providing a sample possibly containing one or more targets as well as possibly other molecules that are not targets, providing one or more probe sets each comprising either: a) one or more HCR initiator-labeled probes, or b) one or more probe units each comprising two or more HCR fractional initiator probes, providing one or more HCR amplifiers (each labeled with one or more haptens), providing one or more anti-haptens labeled with one or more reporter entities, wherein the reporter entity is an enzyme that mediates CARD, providing one or more CARD-substrates leading to deposition of one or more reporters, and detecting one or more signals from one or more reporters.

Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs and/or cDNAs.

Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs and/or cDNAs.

A method for spatially tagging nucleic acids of a biological specimen, including steps of (a) providing a solid support comprising different nucleic acid probes that are randomly located on the solid support, wherein the different nucleic acid probes each includes a barcode sequence that differs from the barcode sequence of other randomly located probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequences on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located probes; (d) hybridizing the randomly located probes to target nucleic acids from portions of the biological specimen; and (e) modifying the randomly located probes that are hybridized to the target nucleic acids, thereby producing modified probes that include the barcode sequences and a target specific modification, thereby spatially tagging the nucleic acids of the biological specimen.

A method for spatially tagging nucleic acids of a biological specimen, including steps of (a) providing a solid support comprising different nucleic acid probes that are randomly located on the solid support, wherein the different nucleic acid probes each includes a barcode sequence that differs from the barcode sequence of other randomly located probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequences on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located probes; (d) hybridizing the randomly located probes to target nucleic acids from portions of the biological specimen; and (e) modifying the randomly located probes that are hybridized to the target nucleic acids, thereby producing modified probes that include the barcode sequences and a target specific modification, thereby spatially tagging the nucleic acids of the biological specimen.

A method of simultaneously detecting target nucleic acids and target proteins in a biological sample, comprising treating the biological sample with a crosslinking agent, that is after incubating it with a primary antibody that detects the target proteins and prior to detecting the target nucleic acids by in situ hybridization.

A method of simultaneously detecting target nucleic acids and target proteins in a biological sample, comprising treating the biological sample with a crosslinking agent, that is after incubating it with a primary antibody that detects the target proteins and prior to detecting the target nucleic acids by in situ hybridization.

This invention relates to the preparation of nucleic acid samples for analysis. The invention may be particularly useful for single stranded samples. Embodiments of the invention involve the attachment of double stranded or hairpin oligonucleotides using template independent polymerase enzymes in the preparation of nucleic acid sequencing libraries.

This invention relates to the preparation of nucleic acid samples for analysis. The invention may be particularly useful for single stranded samples. Embodiments of the invention involve the attachment of double stranded or hairpin oligonucleotides using template independent polymerase enzymes in the preparation of nucleic acid sequencing libraries.

Provided herein are methods and systems for detection of nucleic acids for single cell samples. As part of the detection, a unique step of normalization of different single cell samples is included. One embodiment of the method includes i) selecting one or more target nucleic acid sequence of interest in an individual cell, where the target nucleic acid sequence is complementary to a nucleic acid in a cell; ii) providing a sample having a plurality of individual single cells and encapsulating one or more individual cell(s); iii) providing a sample normalization component to one or more encapsulated cell, where the normalization component comprises an exogenous nucleic acid having a known sequence; iv) providing nucleic acid primers for the target nucleic acid and the exogenous nucleic acid; v) providing a protease to each encapsulated cell and incubating the encapsulated cell with the protease in the drop to produce a cell lysate; vi) performing a nucleic acid amplification reaction to form an amplification product from the nucleic acid of a single cell, where the amplification product comprise amplicons of one or more target nucleic acid sequence and an amplicon for the exogenous nucleic acid; and vii) comparing the amplification products from the target amplicons and the exogenous nucleic acid amplicons and determining the copy number or sequence of the target nucleic acid in a single cell.