Production of high-quality proximity ligation libraries, using two restriction enzymes.
You would want to use the Arima HiC kit when:
- You have a draft genome you want to polish
- You want to identify topologically associated domains (TADs)
- You have limited available sample material
- Your organism has a complex or large genome
- Sample amount:
- Animal tissues: 50 to 200 mg of non-fatty soft tissue (muscle, internal organs)
- Blood: 400 μl of mammalian blood. 10 μl of blood if nucleated red cells (birds, reptiles, fishes)
- Cells: 1×10ˆ6 to 5×10ˆ6 pelleted cells from cell culture or sorted cells
- Low input (for vertebrates):
- Loop calling from as few as 50,000 cells
- TAD calling from as few as 10,000 cells
- A/B compartment calling from as few as 5,000 cells
- Sample storage:
- Tissues: Snap-frozen in liquid nitrogen (liqN2). Not ideal but accepted: preserved on ethanol or RNAlater.
- Blood: Snap-frozen in liquid nitrogen or dry ice. Do not use lysis buffers, detergents or ethanol to preserve the blood.
- Cells: Snap-frozen pellet in liquid nitrogen. If the cells come from cell culture, wash with 1X PBS 3 times after trypsinization, pellet by centrifugation, remove PBS and freeze in liquid nitrogen.
- Sample extraction method: No extraction is needed, as the prep starts directly from tissue.
How to evaluate the sample quality
The most important aspect in ensuring high sample quality is keeping the tissue frozen, it may not be re-thawed once it has been frozen.
For tissue grinding, the sample should be deposited in a pre-cooled mortar. Add some liquid nitrogen (liqN2), make sure the sample is completely frozen. Grind the sample using the pestle until it resembles a flour-like powder. Transfer using a pre-cooled spatula to a pre-cooled tube. The sample should remain frozen through the whole grinding and transfer process. Add more liqN2 if you notice the sample is melting during grinding. Use liqN2 to pre-cool mortar, spatulas, and tubes. If possible, keep the mortar on dry ice during the whole procedure.
If you can not carry out these steps, or your samples do not meet the requirements, please contact us.
What we do with your samples
Once your samples arrive at NGI, we start by performing a reception control step in which we make sure the sample meets our requirements.
If the samples fail this quality control step, we will contact you to discuss possible options. In the case of Hi-C projects, the primary RC consists of the quantification of the available tissue. During the protocol, there are a number of additional QC steps. If any of those steps fail, we will contact you.
If the samples pass the reception control, we will inform you and the samples will be queued for library prep.
The library preparation consists of 5 stages:
- Sample preparation and cross-linking
Sample quantification and aliquoting if necessary. Chromatin is fixed in place using formaldehyde.
- Lysate quantification
Chromatin is then released by lysing the cells. In this step, the amount of chromatin obtained, as well as the degree of digestion, is assessed to ensure the success of the library prep.
- Digestion The cross-linked chromatin is digested using two restriction enzymes.
- Proximity ligation
End-polishing, ligation of a biotinylated oligonucleotide bridge. Intra-aggregate ligation to capture contacts is performed, followed by cross-link reversal and DNA purification.
- Library preparation
End repair, adapter ligation, and purification steps result in the template for the final stage.
- Ligation capture and amplification
A streptavidin enrichment step allows capturing the products from the proximity ligation step. Indices are added by PCR and a bead purification and size selection yield Illumina compatible libraries.
Library QC and sequencing
In this step, we evaluate the library yield and determine the size distribution of the libraries. We will inform you of the QC status of each sample. Once the libraries have passed this QC step, they are queued for sequencing.
The sequencing will be carried out following the setup stated in the agreement.
The libraries will contain information about which parts of the genome were physically proximal in the nuclei of the cells or tissue used to generate the library. This information can be used to build contact maps, after aligning the reads to the reference genome, or to scaffold a genome assembly into chromosome or near chromosome-scale scaffolds.
Arima-HiC data can be used to study 3D genome conformation by processing it using publicly available tools such as Juicer (Durand, 2016a) or Hi-C Pro (Servant, 2015). Genome organizational features such as compartments, TADs, and loops can be identified and visualized using tools such as Juicebox (Durand, 2016b). Arima-HiC data can be mapped to contigs using our mapping pipeline (https://github.com/ArimaGenomics) or Juicer, and the contigs can then be scaffolded using tools such as SALSA (Ghurye, 2019) or 3D-DNA (Dudchenko, 2017).
Scaffolding genome assemblies:
- NGI runs 3D-DNA or SALSA2 for genome assembly scaffolding
TAD calling and differential contact screens
- NGI is currently exploring software for this application