May 04, 2026
Version 2
Library Preparation using the Arima Library Prep Module V.2
This protocol is a draft, published without a DOI.
- Shadi Melnyk1,
- Allyson Whittaker1,
- Jon Belton1,
- Andrew Kao1,
- Arthur Bautista1,
- Andrea Hart Liabotis1
- 1Arima Genomics
Protocol Citation: Shadi Melnyk, Allyson Whittaker, Jon Belton, Andrew Kao, Arthur Bautista, Andrea Hart Liabotis 2026. Library Preparation using the Arima Library Prep Module. 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 and most up-to-date protocol. Formatting revisions pending.
Created: May 04, 2026
Last Modified: May 04, 2026
Protocol Integer ID: 316328
Keywords: Library Preparation, Arima Library Prep Module, Genome Wide HiC Kit, arima library prep module this protocol, arima library prep module, library preparation, protocol detail, protocol, arima library prep module reagent, hic library, libraries while dna, library amplification reagent, adapter ligation protocol, pcr amplification of the bead, final arima, pcr amplification, library, index pcr primer, dna fragmentation, dna, biotin enrichment, dna size selection, bound arima, protocol yield
Abstract
This protocol details the library preparation using the Arima library prep module. Library preparation begins with DNA fragmentation, DNA size selection, and biotin enrichment. Afterward, the Arima Library Prep Module reagents are used in a custom end-repair, dA-tailing, and adapter ligation protocol. This protocol yields construct libraries while DNA is bound to T1 Beads. The final step is PCR amplification of the bead-bound Arima-HiC Library using the library amplification reagents and index PCR primers from the Arima Library Prep Module, producing the final Arima-HiC Library.
Guidelines
Workflow Overview
Figure 1. Arima-HiC+ workflow
Materials
- Arima-HiC+ Kit (PN A510008)
- Arima Library Prep Module (PN A303011)
- Deionized Water (Fisher Scientific‱ Cat # LC267402)
- 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
- Thermomixer
- Gel Electrophoresis System (e.g. Bioanalyzer‱ , TapeStation‱ , FlashGel‱ , etc.)
Troubleshooting
Problem
Adapter or Primer Dimers
Solution
An additional 0.8X size selection can be used if adapter/primer dimers are present in the final libraries.
Before start
Library Preparation :
DNA Fragmentation
The output of the Arima-HiC Protocol is large proximally-ligated DNA molecules. These large DNA molecules must be fragmented using mechanical methods to limit sequence bias, and then prepared as a sequencing library that is compatible with Illumina‱ sequencing instruments. Covaris‱ instruments are recommended for mechanical fragmentation of DNA, although Diagenode‱ instruments have also been tested and yield comparable results. DNA should be fragmented in 100µL of Elution Buffer (Arima HiC+, Box A). Some Covaris‱ protocols recommend DNA fragmentation in 130µL, but 100µL must be used for DNA fragmentation in the Arima-HiC library preparation protocol. It is recommended to fragment 1,500ng of DNA per sample, or up to 5µg (depending on the DNA fragmentation instrument manufacturer's recommendations). For certain applications, less than 750ng of DNA could be used.
DNA Size Selection
Fragmented DNA must be size-selected to have a size distribution between ~200 – 600bp. This workflow can be performed in microfuge tubes as well as most PCR tubes and PCR plates. Ensure that your tubes or plates can hold up to 200µL of sample volume. Also, ensure that you have a magnetic rack that fits your choice of sample tube/plate.
Biotin Enrichment
This workflow can be performed in microfuge tubes as well as most PCR tubes and PCR plates. Ensure that your tubes or plates can hold up to 230µL of sample volume. Also, ensure that you have a magnetic rack that fits your choice of sample tube/plate. Set your thermal device (thermal cycler or thermomixer) to hold at 55°C.
Library Preparation of Enriched Ligation Products
This workflow can be performed in microfuge tubes as well as most PCR tubes and PCR plates. Ensure that your tubes or plates can hold up to 200µL of sample volume. Also, ensure that you have a magnetic rack that fits your choice of sample tube/plate. Set your thermal device (thermal cycler or thermomixer) to hold at 55°C.
Library Preparation
Input: Proximally Ligated DNA generated using the Arima HiC+ Kit (PN A510008) or Arima High Coverage HiC Kit (PN A101030/A101031)
Output: Arima-HiC library ready for sequencing
Library Preparation - DNA Fragmentation
If necessary, add Elution Buffer to bring the sample volume to 100 µL . Do not exceed 100 µL of volume for DNA fragmentation.
Fragment DNA to obtain an average fragment size of ~400bp.
Please use the DNA fragmentation instrument manufacturer's default settings for obtaining a target fragment size of ~400bp.
For example, Covaris® publishes optimal DNA fragmentation Power, Duty Factor, Cycles per Burst, and Time for obtaining a target fragment size of 400bp.
Note
For High Coverage HiC samples, fragment DNA to obtain an average fragment size of ~550-600bp.
Exemplary Covaris E220 settings are noted below for obtaining a target fragment size of ~400bp.
Table 14. Fragmentation Settings for Covaris E220
Samples may be stored at -20 °C for up to 3 days.
Recommended QC before proceeding: Run an aliquot of fragmented DNA on a gel electrophoresis system (e.g., Bioanalyzer®, TapeStation®) to confirm an appropriate fragment size distribution centered around 400bp.
Library Preparation - DNA Size Selection
18m
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 T1 Beads provided in the Arima Library Prep. Module. For the ethanol washes performed below, use sufficient 80% ethanol to fully submerge the magnetized beads.
Transfer fragmented DNA sample from fragmentation tube to either a microfuge tube, PCR tube, or PCR plate. If necessary, add Elution Buffer to bring sample volume to 100 µL
Add 100 µL of DNA Purification Beads, mix thoroughly by pipetting, and incubate at Room temperature for 00:05:00 .
5m
Place sample against magnet and incubate until solution is clear.
Discard supernatant. While sample is still against magnet, add 200 µL of 80% ethanol, and incubate at Room temperature for 00:01:00 .
1m
Discard supernatant. While sample is still against magnet, add 200 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 00:01:00 to air-dry the beads.
1m
Remove the sample from magnet, resuspend beads in 30 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 sample tube or well of a PCR plate.
Quantify sample using Qubit®. Record this value.
Samples may be stored at -20 °C for up to 3 days.
Library Preparation - Biotin Enrichment
24m 10s
Note
T1 Beads used directly below are from the Arima Library Prep Module. They should not be mistaken for and are NOT interchangeable with the Arima-HiC+ Enrichment Beads nor the Arima-HiC+ QC Beads
Mix T1 Beads very well before using, making sure that the solution is homogenous and there is nothing sticking to the bottom of the bottle.
Add 12.5 µL of T1 Beads from the Arima Library Prep Box C into a well of a strip tube for each sample.
Wash the T1 Beads in each tube by adding 200 µL of Binding Buffer.
Mix by pipetting up and down 20 times, cap the tubes, and vortex at high speed for 00:00:05 - 00:00:10 .
10s
Place tubes against a magnet and incubate 00:05:00 or until solution is clear.
5m
Discard supernatant and remove the tube from magnet.
Repeat steps 18 – 21 two more times for a total of three washes.
Resuspend beads in 200 µL of Binding Buffer.
Transfer 200 ng of size-selected DNA into a new microfuge tube, PCR tube, or well of a PCR plate. If necessary, add Elution Buffer to bring sample volume to 30 µL
Note
Biotin enrichment and subsequent library preparation have been optimized to deliver peak performance for DNA inputs of 200ng. Using 200ng of DNA input has been shown to build libraries with sufficient complexity for up to 600M read-pairs of sequence data. If the amount of DNA is less than 200ng, add the entire amount.
Add 200 µL of washed T1 Beads in Binding Buffer, 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 . Set lid temperature to 85 °C .
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 Set lid temperature to 85 °C .
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 50 µL of Deionized / Nuclease-free Water.
Library Preparation of Enriched Ligation products - End Repair
Thaw reagents and mix reagents according to Table 15.
Table 15. Thawing and Mixing Instructions for End Repair
Note
Thaw ligation buffer and vortex on high to make sure homogenous (buffer is highly viscous).
Prepare Ligation master mix to allow equilibration to Room temperature before use (see Table 16, which includes 12.5% master mix overage for 8 reactions).
Table 16. Ligation Master Mix Worksheet
Note
Ligation Master Mix will be used in Step 45 below (After End Repair and dA Tailing)
Keep Ligation Master Mix at Room temperature for 00:30:00 - 00:45:00 before use.
Vortex thawed vial of End Repair-A Tailing Buffer for 00:00:15 - continue vortexing until no solids are observed.
15s
Prepare End Repair/dA-Tailing master mix by combining reagents as listed in Table 17 below, mix well and spin down.
Table 17. End Repair/dA Tailing Master Mix Worksheet
Add 20 µL of the End Repair/dA-Tailing master mix to each sample containing 50 µL of Bead bound HiC library. Mix well.
Program thermal cycler for End Repair and dA-Tailing using the parameters in Table 18. Set reaction volume for 70 µL , and the heated lid to 85 °C , and press start. Total run time is approx. 00:30:00 .
Table 18. End Repair and dA-Tailing Thermal Cycler Program
30m
Library Preparation of Enriched Ligation products - Adapter Ligation
32m
Once thermal cycler has reached 4 °C hold step, transfer samples to On ice while preparing the ligation reaction.
Add 25 µL of room temperature Ligation Master Mix to the 70 µL of bead bound, end repaired and dA-tailed HiC library. Mix well.
Add 5 µL of Adaptor Oligo Mix to each sample. Mix well.
Briefly spin tubes with the bead-bound HiC library, Ligation master mix, and Adaptor Oligo Mix.
Program the thermal cycler for the ligation step with the program specified in Table 19 below. Set the reaction volume to 100 µL and the heated lid to 85 °C , and press start. Total time is approx. 00:30:00 .
Table 19. Adapter Ligation Thermal Cycler Program
30m
After the “Ligation” program completes, remove the samples from the thermocycler and quick spin the tubes to remove any liquid from the caps.
Magnetize beads until liquid is clear. Remove and discard supernatant.
Resuspend beads in 200 µL Wash Buffer. Mix by pipetting. Incubate at 55 °C for 00:02:00 . Set lid temperature to 85 °C .
2m
Magnetize beads until liquid is clear. Remove and discard supernatant.
Resuspend beads in 100 µL Elution Buffer.
Magnetize beads until liquid is clear. Remove and discard supernatant.
Resuspend the beads in 34 µL of Deionized Water and proceed immediately to Library Amplification below.
Library Preparation of Enriched Ligation products - Amplification of Adaptor-Ligated HiC Library and Sample Indexing
Thaw and mix the reagents according to the Table 20 below and keep On ice .
Table 20. Thawing and Mixing Instructions
Thaw only the index primers needed for experiment to minimize freeze-thaw cycles.
Determine the unique index pair assignment for each sample using Table 21 as a reference.
Table 21. Index Pairs included with the Arima Library Prep Module
Prepare appropriate volume of PCR reaction mix in Table 22 below. Mix well.
Table 22. PCR Reaction Mix
Add 11 µL of the PCR reaction mixture prepared from the table above to 34 µL of Adaptor Ligated Bead Bound HiC Library
Add 5 µL of the appropriate, unique, Index Primer Pair to each sample. Make sure to take note of which index was used with each sample.
Program thermal cycle according to the settings in Table 23.
Table 23. Library Amplification Thermal Cycle Program
Adjust PCR cycles according to recommendations in Table 24.
Table 24. PCR Number Cycle Recommendations
If you are working with <50ng of material, please contact [email protected]
Note
For High Coverage HiC samples, use 12 PCR cycles for DNA inputs between 100ng - 200ng.
Place the PCR reaction in the thermocycler and press play.
Note
The cycle number entered into the thermocycler is typically X-1 (e.g., 10 cycles would be input as return to step 2, 9X). Set Lid Temp to 105°C and the reaction volume to 50µL.
Library Preparation of Enriched Ligation products - Purify Amplified Library with Purification Beads
12m
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 Library Prep kit.
Add 50 µL of DNA Purification Beads to each 50 µL Indexed sample. Mix well.
Incubate for 00:05:00 at Room temperature .
5m
Place sample against magnet and incubate until solution is clear.
Discard supernatant. While sample is still against magnet, add 200 µL of 80% ethanol, and incubate at Room temperature for 00:01:00 .
1m
Discard supernatant. While sample is still against magnet, add 200 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 the sample from magnet, resuspend beads in 15 µL of Deionized / Nuclease-free Water, and incubate at Room temperature for 00:05:00 .
5m
Place sample against magnet and incubate until solution is clear.
Remove purified and complete HiC library and transfer to a fresh PCR strip tube.
Prepare a 1:10 dilution (1 µL of sample with 9 µL of water).
Quantify sample using Qubit® using 1 µL .
Run the sample from the previous step on a gel or other platform to determine the size distribution of the HiC library.
Samples may be stored at -20 °C for up to 6 months.