ExperimentTypein situ Hi-C

   March 28th, 2019 at 3:29pm
Reference Publication
Rao SS et al. (2014) PMID:25497547
Rao SS, Huntley MH, et al., Cell 2014
Approved SOP
4DN Standard in situ Hi-C protocol
The Official 4DN Standard in situ Hi-C protocol adapted from Rao et al. 2014 PMID: 25497547 - with accepted deviations approved by SC 02/21/2017


Experiment Category 
Assay Classification 
3C via Ligation
Experimental Purpose 
DNA-DNA Pairwise Interactions
Raw Files Available 
Reads (fastq) provided by lab

Assay Description

In Situ Hi-C

In situ Hi-C is a method to detect and quantify the pairwise interactions between chromosome regions across the entire genome. It was developed in 2014 as an improvement over the dilution Hi-C method. Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed in situ inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.

The protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. Analysis of the resulting paired-end short read sequences produces a matrix that shows the number of interactions between different DNA regions.

See Rao et al., 2014 for more details.

Image source: Rao et al., 2014, Figure 1A