Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.

Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.

The invention is directed to a method to obtain the spatial location and sequence information of at least a part of a RNA or cDNA strand (006) in a sample comprising the steps a. hybridizing a first detection probe oligonucleotide (204) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the at least one RNA or cDNA strand, wherein the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide (205) comprising 5-100 nucleotides wherein a gap region (206) capable of binding oligonucleotides is created b. filling the gap region (206) in part with 1 to 16 barcode oligonucleotides comprising 4-20 nucleotides, wherein the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample c. partially hybridizing a second detection probe oligonucleotide (204′) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the same or cDNA strand and with the respective other end to the bridge oligonucleotide (205) to create a circular template d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers e. determining the sequence of nucleotides of the rolonies

The invention is directed to a method to obtain the spatial location and sequence information of at least a part of a RNA or cDNA strand (006) in a sample comprising the steps a. hybridizing a first detection probe oligonucleotide (204) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the at least one RNA or cDNA strand, wherein the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide (205) comprising 5-100 nucleotides wherein a gap region (206) capable of binding oligonucleotides is created b. filling the gap region (206) in part with 1 to 16 barcode oligonucleotides comprising 4-20 nucleotides, wherein the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample c. partially hybridizing a second detection probe oligonucleotide (204′) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the same or cDNA strand and with the respective other end to the bridge oligonucleotide (205) to create a circular template d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers e. determining the sequence of nucleotides of the rolonies

The invention is directed to a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the steps a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker b. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomer c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker d. removing sample form surface e. hybridizing at least one oligonucleotide comprising 50-1000 nucleic acids with its 5′ and 3′ ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular template f. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming rolonies g. obtaining the sequence information of the rolonies h. linking the spatial information of the sample with the sequence information of the rolonies.

The invention is directed to a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the steps a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker b. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomer c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker d. removing sample form surface e. hybridizing at least one oligonucleotide comprising 50-1000 nucleic acids with its 5′ and 3′ ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular template f. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming rolonies g. obtaining the sequence information of the rolonies h. linking the spatial information of the sample with the sequence information of the rolonies.

The present disclosure relates to a method of sequencing nascent RNA in a cell. In some embodiments, the nascent RNA is conjugated to DNA using copper-catalyzed azide-alkyne cycloaddition (CuAAC). Methods of the present disclosure can be used to generate genomic libraries of a cell and measure gene expression and enhancer and/or super-enhancer activity.

The present disclosure relates to a method of sequencing nascent RNA in a cell. In some embodiments, the nascent RNA is conjugated to DNA using copper-catalyzed azide-alkyne cycloaddition (CuAAC). Methods of the present disclosure can be used to generate genomic libraries of a cell and measure gene expression and enhancer and/or super-enhancer activity.

Provided herein are methods for preparing biological samples for spatial proteomic analysis, methods of determining a location of a protein analyte in a biological sample, and methods of determining a location of a protein analyte and a nucleic acid analyte in a biological sample.

Provided herein are methods for preparing biological samples for spatial proteomic analysis, methods of determining a location of a protein analyte in a biological sample, and methods of determining a location of a protein analyte and a nucleic acid analyte in a biological sample.