A proximity-ligation protocol using a sequence-independent endonuclease, generating data for TAD identification and scaffolding.
The Omni-C kit from Dovetail allows you to produce high-quality proximity ligation libraries. This protocol increases the genomic coverage and reduces biases when compared with restriction enzyme-based Hi-C preps. You would want to use this protocol when:
- Sample amount:
- Animal tissues: 20 to 40 mg of non-fatty soft tissue (internal organs). If the sample is muscle tissue, please provide at least 50mg.
- Plant tissues: 300 mg of young leaves or non-fibrous tissue
- If stored in EDTA, Heparin, or ACD-A, 1ml of blood.
- If is flash-frozen, 3ml of blood.
- Nucleated: 10 μl of blood if nucleated red cells (birds, reptiles, fishes).
- Recommended: collect the blood and flash-freeze in Liquid Nitrogen. Do not store in any buffers.
- Alternatively, store in EDTA (we still prefer flash-frozen!).
- Cells: 1×10ˆ6 pelleted cells from cell culture or sorted cells
- Low input (for vertebrates):
- 100,000 to 500,000 cells
- 5 mg of any tissue but muscle tissue
- 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. The prep starts directly from tissue.
- Sample Grinding for Tissues: Pre-cool a small mortar and a pestle. This can be done by pouring liquid nitrogen (liqN2) into it and then wait for it to evaporate. If possible, keep the mortar on dry ice during the whole procedure.
Transfer the tissue to the mortar. Carefully add some liqN2 on top of the sample, make sure the sample is completely frozen, wait until the liqN2 is almost gone, and start the grinding. Grind the sample using the pestle until it resembles a baking flour powder. Transfer the powder using a pre-cooled spatula to a pre-cooled 2ml tube. The sample should be always frozen during grinding and transfer. Add more liqN2 if you notice the sample is melting during grinding.
How to evaluate the sample quality
The most important factor in ensuring a high quality sample for Hi-C is avoiding thawing of the tissue sample.
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 be kept frozen during the whole grinding and transfer process. Add more liqN2 if you notice the sample 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 are not able to carry out these steps, or your samples are below the requirements, please contact us.
What we do with your samples
Once your samples arrive at NGI, we start by performing a reception control QC 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.The primary reception control of Hi-C samples involves the quantification of the available tissue. During the protocol, there is 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 aliquotation if necessary. Chromatin is fixed in place using formaldehyde. After cross-linking is stopped, an in situ digestion of the chromatin is performed using an endonuclease enzyme.
- 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.
- 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 the capture of 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 yield and size distribution of the libraries and inform you about 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 expected result is that the sequenced 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 of the samples after aligning the reads to the reference genome or to scaffold a genome assembly into chromosome or near chromosome-scale scaffolds.
Regardless of sample type, Omni-C should generate libraries with high complexity and long-range information, even at low input. When compared to other restriction enzyme-based Hi-C data, we expect to have a more evenly distributed coverage across the genome. This allows for down-stream analyses such as SNP calling and phasing.
The even coverage of the Omni-C data should also make the scaffolding of contigs for genome assembly more accurate since no contig will be missing due to the lack of restriction sites, as may happen with classic Hi-C.
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