Gramc: genome-scale reporter assay method for cis-regulatory modules

A method of constructing a nucleic acid molecule reporter library, comprising:

• (i) isolating a plurality of nucleic acid molecules of a selected size range;
• ligating the plurality of isolated nucleic acid molecules of the selected size range to at least one linear adapter sequence using a ligase, wherein the linear adapter sequence comprises at least two consecutive ribonucleotides flanked by at least one deoxyribonucleotide on the 3′ end, and at least one deoxyribonucleotide on the 5′ end, thereby producing a plurality of circular nucleic acid molecules comprising an insert and an adapter;
• (ii) contacting the plurality of circular nucleic acid molecules comprising an insert and an adapter with an exonuclease under conditions sufficient to remove linear nucleic acid molecules from the plurality of circular nucleic acid molecules;
• (iii) contacting the plurality of circular nucleic acid molecules comprising an insert and an adapter with an endoribonuclease under conditions sufficient to produce a plurality of linear nucleic acid molecules each comprising the at least one deoxyribonucleotide on the 3′ end and the at least one deoxyribonucleotide on the 5′ end, flanking the insert;
• (iv) determining genomic coverage of the plurality of linear nucleic acid molecules comprising the at least one deoxyribonucleotide on the 3′ end and the at least one deoxyribonucleotide on the 5′ end, flanking the insert, comprising:
• (a) selecting at least one genomic region of interest,
• (b) amplifying the plurality of linear nucleic acid molecules comprising the at least one deoxyribonucleotide on the 3′ end and the at least one deoxyribonucleotide on the 5′ end, flanking the insert, and
• (c) determining the whether the selected genomic region is present in the plurality of linear nucleic acid molecules;
• and
• (v) fusing the plurality of linear nucleic acid molecules comprising the at least one deoxyribonucleotide on the 3′ end and the at least one deoxyribonucleotide on the 5′ end, flanking the insert to at least one reporter nucleic acid to produce a plurality of reporter constructs, comprising:
• (a) fusing the plurality of linear nucleic acid molecules comprising the at least one deoxyribonucleotide on the 3′ end and the at least one deoxyribonucleotide on the 5′ end, flanking the insert to a linear vector nucleic acid, thereby producing a plurality of linear vectors;
• (b) contacting each of the plurality of linear vectors with a primer comprising a barcode nucleic acid;
• and
• (c) performing a polymerase chain reaction (PCR), producing a plurality of circular vectors comprising a barcode reporter construct comprising the at least one deoxyribonucleotide on the 3′ end and the at least one deoxyribonucleotide on the 5′ end, flanking the insert and a barcode;
• and
• (d) contacting the plurality of circular vectors comprising the barcode reporter construct with an exonuclease under conditions sufficient to remove linear nucleic acid molecules from the plurality of circular vectors comprising a barcode reporter construct.