ExperimentType DNA SPRITE

   March 28th, 2019 at 6:07pm
Reference Publication
Quinodoz SA et al. (2018) doi:10.1016/j.cell.2018.05.024
Quinodoz SA, Ollikainen N, et al., Cell 2018


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

Assay Description


SPRITE is a method to detect and quantify genome-wide higher-order interactions that occur simultaneously within the same nucleus. It was first published in 2018, and it aims to address certain limitations of proximity ligation and imaging methods. Compared to proximity ligation methods, this technique does not depend on the ligation of spatially close DNA fragments; therefore, it can detect interactions occurring across larger distances in the genome. Additionally, unlike both methods that can only capture simultaneous interactions between a small number of genomic regions (2-3), this technique is able to capture simultaneous interactions between a larger number of genomic regions.

The protocol involves cross-linking the cells to form links between physically adjacent DNA regions and other interacting molecules such as RNA and proteins. Then, the cells are lysed, and a restriction enzyme is used to digest the chromatin into multiple fragments. The cross-linked complexes are coupled to magnetic beads. A split-pool tagging strategy is performed that consists of splitting the cross-linked complexes across a 96 well plate, and ligating a tag sequence unique to each well to each molecule. The wells are then pooled and this process is repeated several times. The molecules located in the same complex will stick together throughout the entire split-pool process, resulting in them having the same barcode combination at the end, while the molecules located in other complexes will have their own distinct barcode combinations. The molecules are sequenced, and all the reads containing the same unique barcode combination are grouped together into a cluster. Initial processing results in the generation of a clusters file where each cluster occupies one line that includes the barcode name and genomic alignments of that cluster. This can be used for additional analysis and to create visualizations. See Quinodoz et. al. Cell 2018 for more details.