May 08, 2026

Arima High Coverage HiC Protocol V.2

This  protocol  is a draft, published without a DOI.
  • Anthony Schmitt1,
  • Jon Belton1,
  • Shadi Melnyk1,
  • Andrea Hart Liabotis1,
  • Arthur Bautista1,
  • Andrew Kao1,
  • Allyson Whittaker1
  • 1Arima Genomics
Andrew Kao
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External link: https://arimagenomics.com/wp-content/files/Arima-High-Coverage-HiC-Kit-User-Guide.pdf
Protocol Citation: Anthony Schmitt, Jon Belton, Shadi Melnyk, Andrea Hart Liabotis, Arthur Bautista, Andrew Kao, Allyson Whittaker 2026. Arima High Coverage HiC Protocol. protocols.io https://dx.doi.org/Version created by Andrew Kao
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Working draft. Typos and formatting checks pending.
Created: May 08, 2026
Last Modified: May 08, 2026
Protocol  Integer ID: 316665
Keywords: Arima hcHiC, Arima High Coverage HiC, Quality control, arima high coverage hic, arima high coverage hic protocol arima high coverage, hic workflow overview, ligated chromatin, separate hic library prep user guide, crosslinked chromatin, dna for biotin, ligated dna, structure of the genome, enriched dna, genome, genome structure, custom library preparation protocol, chromatin, biotinylated nucleotide, dna, ends of dna, restriction enzyme, biotin, available library prep kit
Abstract
Arima High Coverage HiC Workflow Overview
The Arima High Coverage HiC workflow captures the sequence and structure (three-dimensional conformation) of genomes. Chromatin from a sample source (e.g. cell lines) is first crosslinked to preserve the genome structure. The crosslinked chromatin is then digested using a restriction enzyme (RE) cocktail. The 5’-overhangs are then filled in with a biotinylated nucleotide. Next, spatially proximal digested ends of DNA are ligated, capturing the structure of the genome. The proximally-ligated chromatin is then sheared and subjected to a custom library preparation protocol utilizing a pre-validated commercially available library prep kit. A separate HiC Library Prep user guide is provided that contains a custom protocol for enriching the proximally-ligated DNA for biotin and then converting the biotin-enriched DNA to Hi-C libraries.
Guidelines

Figure 1. Arima High Coverage HiC workflow

Sequencing and Data Analysis
Arima High Coverage HiC libraries are sequenced on short-read next-generation sequencing platforms using 2x150 “paired-end” reads. The resulting data is referred to as Arima High Coverage HiC data.

The tools necessary for analyzing Arima High Coverage HiC data depend on the application. For example, for studying 3D genome conformation, Arima High Coverage HiC data can be processed using publicly available tools such as Juicer (Durand, 2016a; https://github.com/aidenlab/juicer/wiki) or Hi-C Pro (Servant, 2015; https://github.com/nservant/HiC-Pro), and genome organizational features such as compartments, TADs, and loops can be identified and visualized using tools such as Juicebox (Durand, 2016b; https://github.com/aidenlab/Juicebox/wiki).

These tools require usage modifications and/or custom input files that are specific to Arima High Coverage HiC data, so please contact Technical Support for assistance in implementing these tools. Additionally, because paired-end reads of Arima High Coverage HiC data can originate from distal sequences along the linear genome; the data captures short- and long-range DNA contiguity information that is valuable for applications such as de novo assembly, genome scaffolding, and haplotype phasing. Arima High Coverage HiC data can be mapped to contigs/unitigs using our mapping pipeline (https://github.com/ArimaGenomics/mapping_pipeline) or Juicer, and then the contigs/unitigs can be scaffolded using tools such as SALSA (Ghurye, 2019;
https://github. com/marbl/SALSA) or 3D-DNA (Dudchenko, 2017; https://github.com/aidenlab/3d-dna). Lastly, because the Arima High Coverage HiC data provides uniform per-base genome coverage while maintaining the highest long-range signal contiguity, it greatly benefits variant discovery, base polishing, scaffolding, and phasing. Please contact Technical Support for more information.
Materials
  • Arima High Coverage HiC Kit (PN A101030/A101031)
  • Deionized Water (Fisher Scientific‱ Cat # LC267402)
  • 1X PBS, pH 7.4 (e.g. Fisher Scientific‱ Cat # 50–842–949)
  • Freshly prepared 80% Ethanol
  • DNA Purification Beads (e.g. Beckman Coulter Cat #A63880)
  • Qubit‱ Fluorometer, dsDNA HS Assay and tubes (Fisher Scientific Cat # Q32851, Q32856)
  • 1.5mL, 15mL and 50mL tubes, including LoBind 1.5mL tubes (e.g. Genesee Cat # 86–923)
  • PCR tubes (e.g. SSIbio‱ Cat # 3247–00) or PCR plates (e.g. Bio-Rad‱ Cat # HSS9641)
  • Magnetic rack compatible with tube choice (e.g. Thermo Fisher Scientific‱ Cat # 12321D)
  • Centrifuge
  • Thermal cycler (if performing parts of Arima-HiC in PCR tubes or PCR plate)
  • Thermomixer
  • Gel Electrophoresis System (e.g. Bioanalyzer‱ , TapeStation‱ , FlashGel‱ , etc.)
Troubleshooting
Problem
QC1 Failure
Solution
1. Increase Conditioning at 62°C from 10 minutes to 20 minutes (This may increase inter-interactions) 2. Increase Digestion (reagents in yellow caps) at 37 °C from 60 minutes to overnight. Complete the incubations at 65 °C and 25 °C as normal the following day 3. Increase Reverse Crosslinking (reagents in purple caps) at 68 °C from 90 minutes to overnight. Recommended for all tissue samples. Not necessary for cells.
Problem
Other
Solution
For any other questions or troubleshooting, please contact [email protected]
Before start
This protocol assumes input material has already been crosslinked. Please refer to the linked protocol for crosslinking instructions or contact [email protected]. The cell or tissue pellet for one Arima High Coverage HiC reaction should occupy no more than 20µL of volume and should be devoid of any residual liquid. If the cell pellet occupies greater than 20µL of volume, aliquot the cells such that the sum of the DNA input from all reactions is between ~500ng-5µg and each cell pellet occupies no more than 20µL of volume, or contact Technical Support for additional guidance. Some of the reaction volumes during incubation steps in thermal cyclers are greater than 100µL. For such volumes, set the reaction volumes on the thermal cycler to 100µL. The volumes have been tested, and no adverse effect on the enzymatic performance of the reactions has been observed.
Arima High Coverage HiC Protocol
6h 2m
Input: Crosslinked cells, tissue, or nuclei containing ~500 ng - 5 µg of DNA Output: Proximally-ligated DNA

Note
Choose to perform either:
Step 1.1: The sample input volume is <20uL and well homogenized.

Step 1.2: The sample input volume is >20uL and is clumpy, sticky, and difficult to pipette (uses 1 additional kit reaction).

Step 1.3: Only if the input sample type is crosslinked nuclei that have been previously purified from tissue.


Resuspend one reaction of pulverized tissue or crosslinked cells in 20 µL of Lysis Buffer in a tube or a well of a PCR plate, and incubate at 4 °C for 00:30:00 .

30m
Resuspend one reaction of pulverized tissue or crosslinked cells in 40 µL of Lysis Buffer in a tube or a well of a PCR plate, and incubate at 4 °C for 00:30:00 . Following the incubation split into 2 reactions and proceed to the next step.

30m
Resuspend one reaction of purified crosslinked nuclei in20 µL of Water in a tube or a well of a PCR plate and procced to the next step.

Add 24 µL of Conditioning Solution, mix gently by pipetting, and incubate at 62 °C for 00:10:00 . If using a thermal cycler, set the lid temperature to 85 °C .
Note
For difficult sample types, this incubation can be held for 00:20:00 . This may increase interchromosomal interactions.


10m
Add 20 µL of Stop Solution 2, mix gently by pipetting, and incubate at 37 °C for 00:15:00 . If using a thermal cycler, set the lid temperature to 85 °C .

15m

Note
Steps 4, 6, 8, and 10 require the addition of several reagents in the same step. These reagents should be combined into master mixes following the master mix tables.

Add 13.5 µL of the digestion master mix containing the reagents in Table 9.
Table 9. Reagent Volumes for Preparing Digestion Master Mix

Mix gently by pipetting, and incubate as follows. If using a thermal cycler, set the lid temperature to 85 °C . Note that there are sequential incubations at different temperatures:
Table 8. Thermal Cycler Program for Digestion

Note
For difficult sample types, incubation at 37 °C can be held Overnight , and complete the incubation at 65 °C for 00:20:00 the following day.

1h 20m
Add 16 µL of fill-in master mix to each sample.

Table 10. Reagent Volumes for Preparing Fill-in Master Mix


Mix gently by pipetting, and incubate at Room temperature for 00:45:00 .

45m
Add 82 µL of ligation master mix containing the following reagents in Table 11.
Table 11. Reagent Volumes for Preparing Ligation Master Mix

Mix gently by pipetting, and incubate at Room temperature for 00:15:00 .

15m
Add 35.5 µL of reverse crosslinking master mix from Table 13.
Table 13. Reagent Volumes for Preparing Reverse Crosslinking Master Mix

Note
Take out Enzyme D from -20°C in advance and leave at RT. Enzyme D should be warmed to RT to help to prevent precipitation in the master mix.

Add 20 µL of Buffer E, mix gently by pipetting up and down and incubate as follows. If using a thermal cycler, set the lid temperature to 85 °C .
Table 12. Thermal Cycler Program for Reverse crosslinking

Note
Overnight incubation at 68 °C must be performed if tissue particles are still readily visible. This overnight incubation must be performed using a thermal cycler with a heated lid set at 85 °C .

* To provide flexibility, this incubation can also be held Overnight at 4 °C . Use a thermal cycler with a heated lid when incubating at 68 °C for longer than 01:30:00 . set at 85 °C
2h
Add 100 µL of DNA Purification Beads, mix thoroughly, and incubate at Room temperature for 00:05:00 .

Note
DNA Purification Beads (e.g. AMPure‱ XP Beads) should be warmed to RT and thoroughly mixed before use. The DNA Purification Beads are a user-supplied reagent and should not be mistaken for the Enrichment Beads or QC Beads provided in the Arima-HiC kit.

5m
Place sample against magnet and incubate until solution is clear.
Discard supernatant. While sample is still against magnet, add 300 µL of 80% ethanol, and incubate at Room temperature for 00:01:00 .

1m
Discard supernatant. While sample is still against magnet, add 300 µL of 80% ethanol, and incubate at Room temperature for 00:01:00 .

1m
Discard supernatant. While sample is still against magnet, incubate beads at Room temperature for 00:03:00 – 00:05:00 to air-dry the beads.

Note
If 2 Arima High Coverage HiC reactions had to be performed due to fatty difficult tissue, elute each Arima High Coverage HiC reaction in 50µL of elution Buffer in step 29 below, and then combine the two samples prior to sample quantification in Step 19.

5m
Remove sample from magnet, resuspend beads thoroughly in 100 µL of Elution Buffer, and incubate at Room temperature for 00:05:00 .
5m
Place sample against magnet, incubate until solution is clear, and transfer supernatant to a new tube.
Quantify sample using Qubit.

Note
If the proximally-ligated DNA yield is less than 275ng, we recommend skipping the Arima-QC1 assay mentioned in Step 20 and described in the following Arima-QC1 Quality Control section.

Transfer 75 ng of sample into a new tube labelled “Arima QC1”, and if needed add elution Buffer to Arima QC1 to bring the volume to 50 µL . The “Arima QC1” sample should now contain 75 ng of proximally-ligated DNA in 50 µL of elution Buffer. Store at -20 °C until use in the following Arima QC1 Quality Control protocol.

Note
Safe Stopping Point: If you are not proceeding to Arima QC 1 or Arima Library Prep, proximally-ligated DNA can be stored at -20 °C up to 14 days.

Arima QC1 Quality Control
19m
Input: Proximally-ligated DNA Output: QC1 measurement

If necessary, thaw the “Arima QC1” samples prepared in the Arima High Coverage HiC Protocol in the previous section.
Add 50 µL of QC Beads, mix thoroughly by pipetting, and incubate at Room temperature for 00:15:00 .

15m
Place sample against magnet, and incubate until solution is clear.
Discard supernatant, and remove sample from magnet.
Wash beads by resuspending in 200 µL of Wash Buffer, and incubate at 55 °C for 00:02:00 .

2m
Place sample against magnet, and incubate until solution is clear.
Discard supernatant, and remove sample from magnet.
Wash beads by resuspending in 200 µL of Wash Buffer, and incubate at 55 °C for 00:02:00 .

2m
Place sample against magnet, and incubate until solution is clear.
Discard supernatant, and remove sample from magnet.
Wash beads by resuspending in 100 µL of elution Buffer.

Place sample against magnet, and incubate until solution is clear.
Discard supernatant, and remove sample from magnet.
Resuspend beads in 7 µL of elution Buffer. Proceed to next step with resuspended beads.

Quantify the total amount of bead-bound DNA using Qubit. Use 2 µL of thoroughly mixed bead-bound DNA for the Qubit assay.

Note
Ensure that the beads are thoroughly resuspended in the elution buffer before taking an aliquot for the Qubit.

Determine the Arima QC1 value by following the Arima High Coverage HiC QC Worksheet. High quality Arima QC1 values are expected to have a value of >15% of the original 75ng input onto the beds, indicating sufficient biotinylation of the HiC DNA. If the Arima QC1 value did not obtain a ‘PASS’ status, please contact Technical Support for troubleshooting assistance.

If Arima QC1 values are >15%, proceed to library prep. After completion of quantification step, the remaining bead-bound DNA can be discarded.