Frequently Asked Questions

SAMPLE SUPPORT
What is the required DNA input amount for the Arima-HiC kit?

The Arima-HiC assay works optimally on cell or tissue samples comprising 3ug DNA. For most human cell lines, this usually corresponds to 500,000 to 1,000,000 cells as input.

Does the Arima-HiC kit support low input samples?

While a cell or tissue sample comprising 3ug of DNA is the recommended input amount for the Arima-HiC assay, it is possible to produce high quality Arima-HiC libraries from much fewer cells (e.g. ~100,000 human cells)

What sample source do you support?

Arima-HiC is compatible with a broad range of sample sources, including human, mammals, non-mammalian animals, and plants. Currently, bacteria samples are not supported.

What sample type do you support?

Arima-HiC is compatible with a broad range of sample types, including:

  • Cultured or primary cells
  • FAC-sorted cells/nuclei
  • Fresh tissues preserved in 1) a cryopreservative buffer containing DMSO and/or glycerol and stored at -80C, 2) ethanol, stored at -80C, and 3) RNAlater, stored at -80C
  • Fresh frozen bulk animal and plant tissues
  • Fresh, non-frozen whole blood
  • Fresh nucleated blood preserved in ethanol, stored at -80C

Arima-HiC is not compatible with

  • Cultured or primary cells (excluding tissues) that have been frozen without prior crosslinking or without the addition of a cell preservation agent
  • Harvested tissue with a long time interval between harvest and freezing.
Will Arima-HiC work on my genome of interest (AT-rich, repetitive, etc.)?

Arima-HiC chemistry is robust and works across a variety of genome compositions. If your genome of interest has unique properties, Arima can run an in silico analysis to inform Arima-HiC assay compatibility.

ARIMA-HIC WORKFLOW
How many reactions are in a kit?

Each Arima Hi-C kit contains 8 reactions. One reaction is typically sufficient for the generation of up to 800M-1B raw read-pairs. More precise estimates of library complexity can be determined from the Arima-QC2 assay as outlined in our User Guide.

How long does it take to complete the Arima-HiC protocol?

The Arima-HiC protocol is a fast, user-friendly workflow that takes on average 6 hours total time with 1 hour hands-on time. The Arima-HiC protocol is followed by library preparation to generate Arima-HiC libraries that are ready for Illumina sequencing.

Is the Arima-HiC kit amenable to Capture-HiC?

Yes, the Arima-HiC kit can be used for Capture-HiC studies. For help with Capture-HiC probe design or more information on compatible capture hybridization protocols, please contact Technical Support.

Is the Arima-HiC kit compatible with HiChIP or PLAC-Seq?

Yes, the Arima-HiC kit can be used for HiChIP and PLAC-Seq studies.

I already have cells that have been previously crosslinked and frozen. Would these be suitable as input to Arima-HiC Protocol?

Yes. Arima recommends crosslinking using 2% formaldehyde (see our User Guide for more details). Crosslinking with different strengths of formaldehyde (e.g. 1%) has also worked with comparable performance. However, we do not recommend using <1% formaldehyde.

What library preparation kits are compatible with the Arima-HiC kit?

Arima currently provides pre-validated custom library prep user guides for KAPA HyperPrep, Illumina TruSeq, and Swift Accel-NGS 2S Plus.

The DNA shearing protocol indicates that proximally-ligated DNA should be sheared in 100ul of volume, but the Covaris user manual indicates that 130ul of volume should be used for shearing in microtubes, which should I use?

We strongly recommend following the Arima user guide and use 100ul for shearing. We have validated that shearing in 100ul of volume in Covaris microtubes produces comparable results to 130ul. Perhaps more importantly, the DNA Size Selection protocol following DNA shearing uses specific SPRI bead to sample volume ratios for bead-based size selection. If 130ul of volume is used as input to the size selection protocol instead of 100ul, the resulting DNA sizes will be considerably larger than expected and may negatively impact library prep and sequencing performance.

What ancillary equipment is needed to prepare Arima-HiC libraries?

The preparation of Arima-HiC libraries requires a centrifuge for pre-HiC sample prep, a thermomixer or thermal cycler for heated incubations, a Covaris or Diaganode sonicator for DNA shearing, a thermal cycler for PCR, and a Qubit and qPCR machine for DNA quantifications.

QUALITY CONTROL
Does the Arima-HiC kit provide a way to optimize the amount of sample material to use as input to an Arima-HiC reaction?

The Arima-HiC workflow has one “pre-HiC” quality control assay used to optimize the input material into an Arima-HiC reaction. This protocol is called “Determining Input Amount” and can be found as a section preceding the Arima-HiC Protocol in the Cell Line, Plant Tissue, and Animal Tissue User Guides. The Arima-HiC kit supplies enough reagent to perform this protocol on 8 samples, one for each reaction in the Arima-HiC kit.

What QC steps are involved in ensuring high-quality and high-complexity Arima-HiC libraries?

The Arima-HiC workflow has two pre-sequencing quality control steps. Arima-QC1 is to used to assess the quality of proximally-ligated DNA produced by the Arima-HiC protocol, and Arima-QC2 is used to assess the overall experimental quality of the Arima-HiC workflow following library preparation but prior to library amplification. A QC worksheet is provided with the Arima-HiC User Guide to help calculate these QC values. Optional shallow Illumina sequencing can also be performed as a final QC step prior to deeper sequencing.

ANALYSIS
What read length should I sequence Arima-HiC libraries?

Arima-HiC libraries can be sequenced using a variety of read lengths offered by Illumina sequencing instruments. In our experience, optimal results are obtained using 2x150bp because longer reads afford higher read mappability, however shorter reads (e.g. 2x36bp) can also be used.

Does Arima Genomics provide their own analysis software?

No, Arima Genomics currently does not provide our own software for downstream analysis of sequenced Arima-HiC libraries. For the generation of HiC contact maps and identification and annotation of DNA loops and topological domains, Arima provides support for many open source tools, including Juicer, HiC-Pro, HOMER, HiCUP, and HiC-Bench. For the scaffolding of genomes and de novo assembly applications, initial data processing can be performed using some of the aforementioned tools, as well as the mapping pipeline found on our GitHub page, and we support the contig scaffolding program SALSA.

Are there any program or file modifications that I will need to begin my data analysis?

For several open-source HiC data analysis tools, you will need to have knowledge of the restriction enzyme cut site motifs and/or genomic locations, as well as the possible ligation junction motifs produced by the Arima-HiC chemistry. This information is commonly used for read trimming and downstream HiC data processing. The Arima-HiC chemistry uses restriction enzymes that digest chromatin at ^GATC and G^ANTC, where N can be any of the 4 genomic bases. Our multiple restriction enzyme chemistry produces the following possible ligation junction motifs: GATCGATC, GANTGATC, GANTANTC, GATCANTC. Please contact Technical Support for more information about how to appropriately implement open-source HiC data analysis tools with respect to our HiC chemistry. We will also be happy to share a link download cut site location files for mouse and human genome builds or help you generate custom cut site location files for your genome of interest.

Will Arima Genomics provide technical support during data analysis?

Yes, Arima Genomics provides comprehensive technical support for both the experimental and analysis portions of Arima-HiC experiments. With respect to data analysis support, the extent of our support is helping users appropriately implement open-source data analysis and visualization tools, and assisting with the quality evaluation of the Arima-HiC data. Arima will also run customers down-sampled Arima-HiC data through internal QC pipelines and provide an assessment of the data quality.