May 04, 2026

Hi-C Protocol Using Arima-HiC+ Kit

This  protocol  is a draft, published without a DOI.
  • Anthony Schmitt1,
  • Jon Belton1,
  • Shadi Melnyk1,
  • Andrea Hart Liabotis1,
  • Allyson Whittaker1,
  • Andrew Kao1
  • 1Arima Genomics
Andrew Kao
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External link: https://arimagenomics.com/wp-content/files/Arima-HiC-Kit-User-Guide-A160509-v2.pdf
Protocol Citation: Anthony Schmitt, Jon Belton, Shadi Melnyk, Andrea Hart Liabotis, Allyson Whittaker, Andrew Kao 2026. Hi-C Protocol Using Arima-HiC+ Kit. protocols.io https://dx.doi.org/
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 and most up-to-date protocol. Revisions for typos and formatting pending.
Created: October 23, 2025
Last Modified: May 04, 2026
Protocol  Integer ID: 316318
Keywords: Arima-QC1, Hi-C, Arima-HiC+, hic data, sequencing, proximity ligation, 3D genomics, crosslinked chromatin, epigenetics , hic, hic protocol, ligated chromatin, hic data, hic data with juicer, hic library, hic workflow, ligated chromatin, dna for biotin, separate hic library prep user guide, crosslinked chromatin, ligated dna, genome, 3d genome conformation, studying 3d genome conformation, structure of the genome, genome structure, biotinylated nucleotide, genome organizational feature, chromatin, enriched dna, sequencing, custom library preparation protocol, arima hosts these file, genome organizational features such as compartment, biotin, processing arima, arimahic data, ends of dna, restriction enzyme, custom arima, dna, analyzing arimahic data, using arima, arima host, several minor modifications to the analysis pipeline, hg19
Abstract
Hi-C Workflow Overview
The Arima-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.

Sequencing and Data Analysis
Arima-HiC libraries are sequenced via Illumina‱ sequencers in “paired-end” mode. The resulting data is referred to as Arima-HiC data. The tools necessary for analyzing ArimaHiC data depend on the application. For example, for studying 3D genome conformation, we recommend processing Arima-HiC data with Juicer (Durand, 2016a). Genome organizational features such as compartments, TADs, and loops can be identified using Juicer tools and visualized using tools such as Juicebox (Durand, 2016b). To accommodate your Arima-HiC data, you will need to make several minor modifications to the analysis pipeline. You will need to provide a custom Arima-HiC-specific restriction site file. In the Juicer version 1.6, this is the -y parameter in the juicer.sh command line script. Arima hosts these files for mm9, mm10, hg19, and hg38 on:
ftp://ftp-arimagenomics.sdsc.edu/pub/JUICER_CUTSITE_FILES.
Guidelines
Workflow Overview

Figure 1. Arima-HiC+ workflow

Materials

  • Arima-HiC+ Kit (Cat # A510008)
  • 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 cell
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-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-HiC Protocol
5h 27m
Input: Crosslinked cells or tissue containing ~500 ng - 5 µg of DNA Output: Proximally-ligated DNA

Note
Choose to perform either Step 1.1 if the input sample type is crosslinked cells, or Step 1.2 only if the input sample type is crosslinked nuclei that have been previously purified from cells.

Resuspend one reaction of 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 purified crosslinked nuclei in 20 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 12 µL of the digestion master mix from 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 from Table 10.
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 from 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

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


Add 20 µL of Buffer E, mix gently by pipetting, 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.


* 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 .



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.

Remove sample from magnet, resuspend beads thoroughly in 100µL 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 the 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”. 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 or proceed directly to Arima-QC1 Quality Control protocol.

Store all remaining samples at -20 °C until ready to proceed to library preparation following an accompanying Arima-HiC Library Preparation user guide.

Arima-QC1 Quality Control
19m
If necessary, thaw the “Arima-QC1” samples prepared during Step 20 of the Arima-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.


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

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

Determine the Arima-QC1 value by following the Arima-HiC QC Worksheet. High-quality Arima-QC1 values are expected to be >15%. If the Arima-QC1 value did not obtain a ‘PASS’ status, please contact Technical Support for troubleshooting assistance.