Disclosed herein are methods, compositions and systems for the interrogation of macromolecules, more particularly for preparation of isolated single macromolecules for subsequent processing of specific regions of interest within said macromolecule based on an analysis of the molecule's physical map. The disclosure is further related to the controlled segmentation of long nucleic acid parent molecules into smaller child molecules in a targeted manner such that further processing on said children may be performed with the knowledge of their origin within the parent, in a controlled environment enabled by purposefully designed microfluidic devices. Also disclosed is binding of regional specific barcodes along the length of a long nucleic acid molecule such that upon cleavage of said molecule into child molecules, the regional origin of the children can be tracked, in a controlled environment enabled by purposefully designed microfluidic devices. Finally, the disclosure is further related to droplet devices and method to control the encapsulation of long nucleic acid molecules or specific subregions thereof into a droplet, and further tracking said droplets with their contents.

The present disclosure is based in part on probes, compositions, methods, and kits for simultaneous, multiplexed spatial detection and quantification of protein and/or nucleic acid expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.--

The present disclosure is based in part on probes, compositions, methods, and kits for simultaneous, multiplexed spatial detection and quantification of protein and/or nucleic acid expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.--

The technology relates in part to methods and compositions for analyzing nucleic acid. In some aspects, the technology relates to methods and compositions for preparing a nucleic acid library from single-stranded nucleic acid fragments.

The technology relates in part to methods and compositions for analyzing nucleic acid. In some aspects, the technology relates to methods and compositions for preparing a nucleic acid library from single-stranded nucleic acid fragments.

The present disclosure provides methods, systems, and kits for processing nucleic acid molecules. A method may comprise providing a template nucleic acid fragment (e.g., within a cell, cell bead, or cell nucleus) within a partition (e.g., a droplet or well) and subjecting the template nucleic acid fragment to one or more processes including a barcoding process and a single primer extension or amplification process. The processed template nucleic acid fragment may then be recovered from the partition and subjected to further amplification to provide material for subsequent sequencing analysis. The methods provided herein may permit simultaneous processing and analysis of both DNA and RNA molecules originating from the same cell, cell bead, or cell nucleus.

The present disclosure provides methods, systems, and kits for processing nucleic acid molecules. A method may comprise providing a template nucleic acid fragment (e.g., within a cell, cell bead, or cell nucleus) within a partition (e.g., a droplet or well) and subjecting the template nucleic acid fragment to one or more processes including a barcoding process and a single primer extension or amplification process. The processed template nucleic acid fragment may then be recovered from the partition and subjected to further amplification to provide material for subsequent sequencing analysis. The methods provided herein may permit simultaneous processing and analysis of both DNA and RNA molecules originating from the same cell, cell bead, or cell nucleus.

The present disclosure relates to compositions, methods, and kits for generating capture probes on a substrate for identifying the location of analytes in a biological sample. In particular, disclosed is a method of generating a spatial array comprising: (a) providing a substrate comprising a plurality of acceptor oligonucleotides, wherein an acceptor oligonucleotide of the plurality of acceptor oligonucleotides comprises a spatial barcode and a first ligation handle, and wherein the 5′ end of the acceptor oligonucleotide is attached to the substrate; (b) providing a plurality of universal splint oligonucleotides, wherein a universal splint oligonucleotide of the plurality of universal splint oligonucleotides comprises a sequence complementary to the first ligation handle and a sequence complementary to a second ligation handle present in a donor oligonucleotide of a plurality of donor oligonucleotides; and (c) ligating the donor oligonucleotide comprising a capture domain to the 3′ end of the acceptor oligonucleotide to generate a capture probe, wherein the universal splint oligonucleotide is hybridized to the first ligation handle and the second ligation handle, thereby generating a spatial array.

The present disclosure relates to compositions, methods, and kits for generating capture probes on a substrate for identifying the location of analytes in a biological sample. In particular, disclosed is a method of generating a spatial array comprising: (a) providing a substrate comprising a plurality of acceptor oligonucleotides, wherein an acceptor oligonucleotide of the plurality of acceptor oligonucleotides comprises a spatial barcode and a first ligation handle, and wherein the 5′ end of the acceptor oligonucleotide is attached to the substrate; (b) providing a plurality of universal splint oligonucleotides, wherein a universal splint oligonucleotide of the plurality of universal splint oligonucleotides comprises a sequence complementary to the first ligation handle and a sequence complementary to a second ligation handle present in a donor oligonucleotide of a plurality of donor oligonucleotides; and (c) ligating the donor oligonucleotide comprising a capture domain to the 3′ end of the acceptor oligonucleotide to generate a capture probe, wherein the universal splint oligonucleotide is hybridized to the first ligation handle and the second ligation handle, thereby generating a spatial array.

Provided herein, among other things, is a method for synthesizing spatially addressed nucleic acid barcodes in or on a cellular sample in situ. In some embodiments, the method may comprise: obtaining a cellular sample comprising nucleic acid molecules that are protected by a reversible terminator, deprotecting the nucleic acid molecules in a set of areas of the sample by selectively applying an external stimulus to the set of areas to produce deprotected nucleic acid molecules in the areas, applying a reversible terminator nucleotide to the cellular sample, resulting in addition of a reversible terminator onto the deprotected nucleic acid molecules, optionally removing any unreacted reversible terminator nucleotide from the sample, and repeating the steps one or more times.