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 invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

Provided are methods for preparation and analysis of nucleic acids. Some embodiments include reverse transcribing the RNA with barcoded primers to produce cDNA while maintaining the DNA in the sample, sequencing the DNA and cDNA together, and differentiating the sequenced DNA and cDNA using the barcode or barcodes of the primers. Some embodiments include analyzing the DNA and cDNA sequences of multiple samples separating reads into k-mers, and comparing the k-mers between samples to identify differential sequences between the sequences of the samples.

Provided are methods for preparation and analysis of nucleic acids. Some embodiments include reverse transcribing the RNA with barcoded primers to produce cDNA while maintaining the DNA in the sample, sequencing the DNA and cDNA together, and differentiating the sequenced DNA and cDNA using the barcode or barcodes of the primers. Some embodiments include analyzing the DNA and cDNA sequences of multiple samples separating reads into k-mers, and comparing the k-mers between samples to identify differential sequences between the sequences of the samples.

Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.

Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.

Provided is a preparation method for a DNA library, comprising a pre-library preparation process, the pre-library preparation process comprising DNA preparation, end repair and 3′ A-tailing, linker connection using an anti-contamination linker, linker connected product purification, pre-library amplification, and amplified pre-library purification. Also provided are a use of the anti-contamination linker in preparing a test kit for DNA library capture, and a method for performing bioinformatic analysis on the DNA library prepared by means of the preparation method of the present invention. The preparation method of the present invention reduces the risk of cross-contamination between samples.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.