In situ Hi-C was performed as described in a previously published protocol (30) with a few modifications. Five million cells were fixed with 1% (v/v) formaldehyde in fresh medium at a concentration of 1 × 106 cells/ml. The reaction was quenched by 0.2 M glycine at room temperature for 5 min. Fixed cells were lysed in cold Hi-C lysis buffer [10 mM tris (pH 8.0) and 10 mM NaCl, 0.2% IGEPAL CA-630 with proteinase inhibitor] for 15 min, followed by a wash with 500 μl of cold Hi-C lysis buffer once. The pellet was gently resuspended in 50 μl of 0.5% SDS and incubated at 62°C for 10 min. Permeabilization was quenched by adding 145 μl of water and 25 μl of 10% Triton X-100 and incubated at 37°C for 15 min. Twenty-five microliters of 10× NEBuffer 2 and 100 U of Mbo I restriction enzyme were added, and chromatin was digested overnight or for at least 2 hours at 37°C, with rotation. Following inactivation of Mbo I at 62°C for 20 min, biotin 2′-deoxyadenosine 5′-triphosphate (dATP) fill-in was performed at 37°C for 1.5 hours with rotation by adding 15 nM biotin dATP, 3′-deoxythymidine 5′-triphosphate, 2′-deoxycytidine 5′-triphosphate, and 2′-deoxyguanosine 5′-triphosphate; 40 U of DNA Pol I; and a large Klenow fragment. In situ ligation was performed at room temperature for 4 hours with slow rotation by adding 900 μl of ligation master mix [120 μl of 10× NEB (New England Biolabs) T4 DNA ligase buffer, 100 μl of 10% Triton X-100, 12 μl of bovine serum albumin (BSA; 10 mg/ml), 5 μl of T4 DNA ligase (400 U/μl), and 663 μl of water]. Following centrifugation at 10,000 rpm for 5 min, the pellet was resuspended in 200 μl of decross-link buffer [50 mM tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS]. Eight microliters of 5 M sodium chloride and 8 μl of proteinase K (10 mg/ml) were added, and reverse cross-linking was performed at 68°C for at least 1.5 hours. Decross-linked DNA was purified by phenol/chloroform extraction. The pellet was dissolved in 200 μl of 1× tris buffer [10 mM tris-HCl (pH 8)] and incubated at 37°C for 15 min to fully dissolve the DNA. The DNA was sheared to a size of 300 to 500 bp by bioruptor with the following setting: high output, 30 s on/30 s off, five times. Sheared DNA was purified by Ampure beads and quantified by Qubit. One hundred fifty microliters of Dynabeads MyOne Streptavidin T1 (10 mg/ml) was used per sample to capture the biotin-labeled fragments. Following biotin pulldown, the DNA was used for library construction.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.