Mapping cell identity determinants in chromatin
View Patent ↗The invention provides materials and methods to identify and analyze in a genome-wide manner the structural determinants of this organization. Next generation sequencing methods, combined with a novel assay and integrated data analysis, are used to map the long-range interactions in chromatin that are involved in the regulation of transcription.
1. A method for processing DNA fragments, comprising:
ligating in a single reaction dN-tailed, cross-linked DNA fragments to four duplex adapters: R1 and R2 recombination adapters and S1 and S2 sequencing adapters, wherein the recombination adapters each contain a site-specific recombinase target site and R1 contains an affinity tag.
2. The method of claim 1 further comprising subsequent steps of:
recombining the target sites with a corresponding site-specific recombinase;
isolating recombined fragments comprising the affinity tag;
amplifying by PCR the recombined fragments comprising the affinity tag with primers complementary to the sequencing adapters; and
sequencing the amplified PCR product.
3. The method of claim 1 further comprising antecedent steps of:
fragmenting cross-linked chromatin into 300-600 bp fragments; and
blunt ending and dN tailing the fragments.
4. The method of claim 1 further comprising antecedent steps of:
fragmenting cross-linked chromatin into 300-600 bp fragments; and
blunt ending and dN tailing the fragments; and subsequent steps of:
recombining the target sites with a corresponding site-specific recombinase;
isolating recombined fragments comprising the affinity tag;
amplifying by PCR the recombined fragments comprising the affinity tag with primers complementary to the sequencing adapters; and
sequencing the amplified PCR product.