The present invention relates to methods for the joint analysis of regulation of gene expression and gene expression in single cells. Provided are methods for obtaining gene expression information for a single nucleus, the methods comprising deriving a DNA library from the genomic DNA in one or more nuclei and deriving an RNA library from the RNA in one or more nuclei, sequencing the molecules in the RNA library and the DNA library, and correlating the RNA library and the DNA library for each of the one or more nuclei.

The present invention relates to methods for the joint analysis of regulation of gene expression and gene expression in single cells. Provided are methods for obtaining gene expression information for a single nucleus, the methods comprising deriving a DNA library from the genomic DNA in one or more nuclei and deriving an RNA library from the RNA in one or more nuclei, sequencing the molecules in the RNA library and the DNA library, and correlating the RNA library and the DNA library for each of the one or more nuclei.

The present invention concerns a method of sequencing immobilized polynucleotides in which beads which are tethered to the solid support are used as labels to identify bases within the polynucleotides. The beads carry sets of probes or bases which can bind to the polynucleotide allowing identification of the target base(s). Identification of the base(s) is achieved through sequential application of different cleavage means specific to different probes/bases carried on the beads. Also provided is an apparatus for performing the method and a kit comprising the apparatus and other components necessary for performing the method.

The present invention concerns a method of sequencing immobilized polynucleotides in which beads which are tethered to the solid support are used as labels to identify bases within the polynucleotides. The beads carry sets of probes or bases which can bind to the polynucleotide allowing identification of the target base(s). Identification of the base(s) is achieved through sequential application of different cleavage means specific to different probes/bases carried on the beads. Also provided is an apparatus for performing the method and a kit comprising the apparatus and other components necessary for performing the method.

Provided herein are novel Class 2 Type II and Type V CRISPR-Cas RNA-guided endonucleases, e.g. Cas9 and Cas12 endonucleases, and systems comprising the same. Provided also are methods of making, and methods of use thereof. Exemplary methods of use include modifying target DNAs and detecting targeting DNAs, useful for therapeutic and diagnostic applications. Some of the diagnostic applications may utilise the collateral nuclease activity of an enzyme bound to a target sequence.

Provided herein are novel Class 2 Type II and Type V CRISPR-Cas RNA-guided endonucleases, e.g. Cas9 and Cas12 endonucleases, and systems comprising the same. Provided also are methods of making, and methods of use thereof. Exemplary methods of use include modifying target DNAs and detecting targeting DNAs, useful for therapeutic and diagnostic applications. Some of the diagnostic applications may utilise the collateral nuclease activity of an enzyme bound to a target sequence.

The invention relates to a nucleic acid comprising at least one methylation-protectable restriction element, the methylation-protectable restriction element comprising: (i) a type IIS restriction enzyme recognition sequence, or a partial type IIS restriction enzyme recognition sequence, that is recognised by a type IIS restriction enzyme that cleaves outside of the recognition sequence; (ii) a DNA methylase recognition sequence that is recognised and methylated by a DNA methylase, wherein the DNA methylase recognition sequence is identical to, or is encompassed within, the type IIS restriction recognition sequence, such that methylation of the nucleic acid by the DNA methylase methylates the type IIS restriction enzyme recognition sequence and protects the nucleic acid from cleavage by the type IIS restriction enzyme; and (iii) a recognition sequence for a sequence-specific DNA-binding protein, wherein the recognition sequence is positioned such that the binding of the sequence-specific DNA-binding protein overlaps with the DNA methylase recognition sequence such that binding of the sequence-specific DNA-binding protein is capable of preventing methylation of the type IIS restriction enzyme recognition sequence by the DNA methylase such that it is not protected from cleavage by the type IIS restriction enzyme. The invention further relates to associated methods of nucleic acid assembly.

The invention relates to a nucleic acid comprising at least one methylation-protectable restriction element, the methylation-protectable restriction element comprising: (i) a type IIS restriction enzyme recognition sequence, or a partial type IIS restriction enzyme recognition sequence, that is recognised by a type IIS restriction enzyme that cleaves outside of the recognition sequence; (ii) a DNA methylase recognition sequence that is recognised and methylated by a DNA methylase, wherein the DNA methylase recognition sequence is identical to, or is encompassed within, the type IIS restriction recognition sequence, such that methylation of the nucleic acid by the DNA methylase methylates the type IIS restriction enzyme recognition sequence and protects the nucleic acid from cleavage by the type IIS restriction enzyme; and (iii) a recognition sequence for a sequence-specific DNA-binding protein, wherein the recognition sequence is positioned such that the binding of the sequence-specific DNA-binding protein overlaps with the DNA methylase recognition sequence such that binding of the sequence-specific DNA-binding protein is capable of preventing methylation of the type IIS restriction enzyme recognition sequence by the DNA methylase such that it is not protected from cleavage by the type IIS restriction enzyme. The invention further relates to associated methods of nucleic acid assembly.

The present invention relates to a method for removing free adapters in a DNA library using a double-stranded nucleic acid molecule and a restriction enzyme, and more specifically, to a method for removing free adapters in a DNA library for next generation sequencing (NGS) using a double-stranded nucleic acid molecule including a type IIs restriction enzyme recognition site and a complementary sequence thereof, and a type IIs restriction enzyme.

The present invention relates to a method for removing free adapters in a DNA library using a double-stranded nucleic acid molecule and a restriction enzyme, and more specifically, to a method for removing free adapters in a DNA library for next generation sequencing (NGS) using a double-stranded nucleic acid molecule including a type IIs restriction enzyme recognition site and a complementary sequence thereof, and a type IIs restriction enzyme.