Method for determination of 3d genome architecture with base pair resolution and further uses thereof

An in situ method for detecting spatial proximity relationships between genomic DNA in in a cell, comprising:

• providing a sample of one or more cells;
• crosslinking the cells with a chemical crosslinker;
• lysing the cells to obtain isolated nuclei;
• permeabilizing the nuclei;
• enzymatically fragmenting the chromatin present in the nuclei;
• performing end repair and/or fill-in on the ends of the chromatin fragments with at least one labeled nucleotide, wherein the labeled nucleotide is capable of being used to isolate the chromatin fragments;
• ligating the repaired and/or filled in ends of the chromatin fragments that are in close physical proximity to create one or more end joined nucleic acid fragments having one or more junctions, wherein the site of the one or more junctions comprises one or more labeled nucleic acids;
• reversing the crosslinking;
• isolating the one or more end joined nucleic acid fragments using the labeled nucleotide;
• and
• sequencing at the one or more junctions of the one or more end joined nucleic acid fragments, thereby detecting spatial proximity relationships between genomic DNA in a cell,
• wherein the steps of enzymatically fragmenting the chromatin present in the nuclei, performing end repair and/or fill-in on the ends of the chromatin fragments with at least one labeled nucleotide, and ligating the repaired and/or filled in ends of the chromatin fragments comprise:
• a. a serial process comprising:
• i. digesting the chromatin with a first restriction enzyme;
• ii. filling in the overhanging ends produced from (i);
• iii. ligating the filled in end of the chromatin fragments from (ii);
• iv. digesting the chromatin fragments from (iii) with a second restriction enzyme;
• v. filling in the overhanging ends produced from (iv);
• and
• vi. ligating the filled in end of the chromatin fragments from (v);
• b. a single-step process comprising:
• i. in a single-step, fragmenting the chromatin present in the cells by contacting the chromatin with two restriction enzymes, filling in one or more overhanging ends of the chromatin fragments, and ligating two or more filled in ends;
• c. a parallel process comprising:
• i. fragmenting the chromatin present in the cell with two restriction enzymes in the same or parallel reactions;
• ii. filling in the overhanging ends from (i), wherein the optional parallel reaction are optionally combined;
• and
• iii. ligating two or more filled ends from (ii), wherein the optional parallel reaction are optionally combined;
• d. a first MNase process comprising:
• i. fragmenting the chromatin using micrococcal nuclease (MNase);
• ii. repairing one or more overhanging ends produced in (i);
• iii. filling in one or more repaired overhanging ends from (ii);
• iv. ligating two or more filled ends from (iii);
• e. a second MNase process comprising:
• i. fragmenting the chromatin present in the cells with MNase;
• ii. in a single step, repairing one or more ends of the chromatin fragments from (i), filling in one or more repaired overhanging ends from (ii), and ligating two or more filled ends from (i);
• or
• f. a third MNase process comprising:
• i. in a single step, fragmenting the chromatin present in the cells with MNase, repairing one or more ends of the chromatin fragments, filling in one or more repaired overhanging ends, and ligating two or more filled ends.