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Sample Support

The Arima-HiC assay works optimally on cell or tissue samples comprising 3 µg of DNA. For most human cell lines, this usually corresponds to 500,000–1,000,000 cells as input. We have successfully tested the Arima-HiC+ protocol starting with an input of 50,000 crosslinked cells, and our customers have published Arima-HiC+ results for 6,000 double-sorted nuclei (Espeso-Gil, et al. Genome Medicine, 2020).

 

While a cell or tissue sample comprising 3 µg 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. ~50,000 human cells or fewer, depending on the application).

When used to perform the Arima-HiC assay, the Arima-HiC+ kit is compatible with a broad range of sample types, including:

  • Cultured or primary cells
  • Fluorescence-activated cell sorted (FACS) cells/nuclei
  • Fresh tissues preserved in 1) a cryopreservative buffer containing DMSO and/or glycerol and stored at -80˚C, 2) ethanol, stored at -80˚C, and 3) RNAlater™, stored at -80˚C
  • Fresh-frozen bulk animal and plant tissues
  • Fresh, non-frozen, whole blood
  • Fresh nucleated blood preserved in ethanol, stored at -80˚C
The Arima-HiC+ kit 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.
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.
The Arima-HiChIP assay works optimally on mammalian cells comprising approximately 12 µg of DNA. For most human cell lines, this usually corresponds to 3,000,000–4,000,000 cells as input.
The Arima-HiChIP protocol is compatible with cultured or primary mammalian cells.

ARIMA-HIC WORKFLOW

Each Arima-HiC+ kit contains 8 reactions. One reaction is typically sufficient for the generation of ~600M raw read-pairs. More precise estimates of library complexity can be determined from the Arima-QC2 assay as outlined in our User Guide.
The Arima-HiChIP protocol involves a 2-day workflow and 1-day library preparation.
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.
Yes, the Arima-HiC+ kit can be used for HiChIP and PLAC-seq studies.
Yes. We recommend 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.
Arima currently provides pre-validated custom library prep user guides for Swift Accel-NGS™ 2S Plus, KAPA HyperPrep™, Illumina TruSeq™ and NEBNext™ Ultra™ II with the Swift protocol strongly preferred for low-input applications. Please contact an Arima representative to inquire about access to the pre-validated user guides.
We strongly recommend following the Arima User Guide and using 100 µL for shearing. We have validated that shearing in 100 µL of volume in Covaris microtubes produces comparable results to shearing in 130 µL. 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 130 µL of volume is used as input to the size selection protocol instead of 100 µL, the resulting DNA sizes will be considerably larger than expected and may negatively impact library prep and sequencing performance.
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 Diagenode™ sonicator for DNA shearing, a thermal cycler for PCR, and a Qubit™ and qPCR machine for DNA quantification.
The requirements for Arima Capture-HiC are the same as for Arima-HiC.
For Capture-HiC, you will follow the Arima-HiC protocol with one modification. This modification is important for generating sufficient material for going into the hybridization step of Capture-HiC and for a quality control sequencing reaction. To generate sufficient material for Capture-HiC hybridization, we recommend splitting the library amplification into 4 reactions. We have found that at higher numbers of PCR cycles the on-bead amplification is less efficient, and by splitting each library into 4 amplification reactions, you will be able to generate sufficient material. The Arima-HiC indexed libraries can be used directly in the Agilent SureSelectXT HS protocol.
We can help you with the bioinformatics to generate probes for your Arima Capture-HiC experiment through our collaborator, Agilent. The probe design with Arima is different for Capture-HiC because we use a multi-enzyme mix, resulting in different cut sites. Arima already has generated probe set sequences in close collaboration with Agilent to look at promoter-specific interaction in human and mice. When using the recommended boosted probe designs with Agilent SureSelectXT HS, a minimum of 500 ng of indexed input DNA is needed. The Arima-HiC indexed libraries can be used directly in the Agilent SureSelectXT HS protocol. We can also help you with designing custom Agilent probe sets for your Arima Capture-HiC experiment in human, mouse, and the reference genomes available in Agilent SureDesign™. For designing a custom Arima Capture-HiC probe set in unsupported or de novo assembled genomes, we can use your target coordinates to generate a target region file that takes into account the Arima cut sites to submit to Agilent Customer Support for probe design. All probe sets will need to be purchased via Agilent.
We recommend using pre-validated custom library prep user guides for Kapa HyperPrep™ or Swift Accel-NGS™ 2S Plus. Please contact an Arima representative to inquire about a pre-validated user guide for your preferred library prep kit.
We recommend using the Accel-NGS™ 2S Plus DNA Library Kit from Swift Biosciences with the Arima-HiChIP kit. 
A 1-mL deep well plate can be used as long as the user can centrifuge at 10,000 x g, and the user can see well enough into the plate to remove the supernatant without disturbing the nuclei pellet.
A lower volume format in PCR plate or strip tube can be used with the following adjustment: 2X with 200 µl of water instead of 1X with 1.5 mL. Plates and tubes must still be centrifuged at 10,000 x g, and the user can see well enough into the plate to remove the supernatant without disturbing the nuclei pellet.
 
We recommend that all buffers be made fresh. The exception is LTE (Tris, low EDTA, TLE) buffer which is commonly stored at room temperature.
 
The samples can be incubated at 25˚C overnight.
Note: In some protocols, samples are incubated at 68˚C overnight to further drive reverse crosslinking.
 
CS Buffer cannot be replaced by Elution Buffer because the SDS concentration is optimized to achieve efficient shearing.
 
We recommend 1000 rpm at 4°C to prevent beads from settling.
Note: A nutator in the fridge may be fine so long as beads do not settle, and the temp is at 4˚C.
 
Arima HiChIP is actively being validated with selected histone and transcription factors. Please contact technical support to determine the current status of your desired antibody.
We recommend the methanol-stabilized formaldehyde.
Note: Using methanol-free formaldehyde might result in reduced crosslinking compared to methanol-stabilized formaldehyde.
 

QUALITY CONTROL

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 “Estimating Input Amount” and can be found as a section preceding the Arima-HiC protocol in all of our 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.
The Arima-HiC workflow has two pre-sequencing, quality control steps. Arima-QC1 is 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.
The Arima-HiChIP workflow has one “pre-HiC” quality control assay used to optimize the input material into an Arima-HiChIP reaction. This protocol is called “Estimating Input Amount” and can be found as a section preceding the Arima-HiChIP protocol in all of our User Guides. The Arima-HiC kit supplies enough reagents to perform this protocol on 8 samples, one for each reaction in the Arima-HiC kit.
The Arima-HiChIP workflow has three pre-sequencing quality control steps and two post-sequencing data QC steps. A QC worksheet is provided with the Arima-HiC+User Guide to help calculate these QC values.  
 

ANALYSIS

Arima-HiC libraries can be sequenced using a variety of read lengths offered by short read sequencing instruments. In our experience, optimal results are obtained using 2 x 150 bp for the majority of applications, because longer reads afford higher read mappability, with the minimum read length being 2 x 36 bp and 2 x 250 bp used for phased de novo assemblies of diploid genomes (Garg, et al. Nature Biotechnology, 2020).
We do 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, we can provide support for a limited number of open-source tools but strongly recommend the use of the Juicer/Juicer Tools pipeline (https://github.com/aidenlab/juicer/wiki). Juicer’s use of the BWA aligner is best suited to map chimeric reads efficiently. Other bowtie-based analysis tools (HiC-Pro, HiCUP, and HiC-Bench) have been successfully used by our customers as well. For genome scaffolding, initial data processing for QC can also be performed using Juicer; we recommend read mapping to be performed via our mapping pipeline found on our GitHub page (https://github.com/ArimaGenomics/mapping_pipeline), followed by our preferred contig scaffolding program, SALSA (https://github.com/marbl/SALSA). Arima High-Coverage HiC is a critical component of the DipAsm assembly algorithm for phased de novo assemblies of diploid genomes (https://github.com/shilpagarg/DipAsm).
For several open-source Hi-C 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 Hi-C 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 Hi-C data analysis tools with respect to Arima-HiC chemistry. We will also be happy to share a link to 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.
Yes, we provide comprehensive technical support for both the experimental and analysis portions of Arima-HiC experiments. We can help users implement our preferred open-source data analysis and visualization tools (Juicer for HiC and MAPS for HiChIP) and assist with the quality evaluation of the Arima-HiC data. We will also run customers’ down-sampled Arima-HiC data through our internal QC pipelines (Juicer for HiC and MAPS for HiChIP) and provide an assessment of the data quality.
For a mammalian genome of 3 Gb, we recommend sequencing two biological replicates per biological condition. For high-resolution analysis of A/B compartments, topologically associating domains (TADs), and chromatin loops, the desired read depth is >600 million paired-end reads for each replicate.  For shallow sequencing used in library QC, we recommend at least 1 million paired-end reads.
We recommend generating up to 1 million on-target Hi-C contacts per 1 MB locus, which will require approximately 10 million reads, depending on the quality of the library. Please contact technical support for more information.
 
We support the Chicago tools found at http://functionalgenecontrol.group/chicago.  For the generation of Hi-C contact maps and identification and annotation of DNA loops and topological domains, we provide support for the use of open-source software, Juicer, and recommend Juicebox for data visualization purposes.


Baitmap and fragment files for mouse and human are available for download. Please contact customer support for details. 
 
We will gladly help you generate the baitmap file for your Arima Capture-HiC experiment. The baitmap file is generated by intersecting the covered region data from your capture probeset with the fragment file of your reference genome/contigs. The fragment file is generated by in silico digestion of your reference genome/contigs with the Arima-specific restriction enzyme cut sites.
We provide detailed recommendations for estimating the optimal Arima-HiChIP sequencing depth to produce robust and reproducible chromatin loop discovery using the MAPS data analysis pipeline. The optimal sequencing depth depends on the number of reads that can be used to identify chromatin loops and the desired resolution of the chromatin looping analysis. For shallow sequencing used in library quality control, we recommend at least 500,000 paired-end reads.

For analysis of Arima HiChIP data, we recommend the Arima-MAPS 2.0 tool. The latest update is available here (https://github.com/ijuric/MAPS), and the original MAPS pipeline is available here (https://github.com/ijuric/MAPS/releases/tag/v1.1.0).  Benchmarking with MAPS, FitHiChIP, HICCUPS, and CriSPRi-validated genomics loops revealed that MAPS has the highest sensitivity with a moderate false-positive rate while minimizing computational time. The MAPS 2.0 pipeline has been updated to include the MACS2 ChIP-seq peak calling algorithm, which allows ChIP-seq peak calling from Arima-HiChIP data as well as improved usability by the expansion of command line options.

A test dataset to verify the Arima-MAPS installation and configuration is available at ftp://ftp-arimagenomics.sdsc.edu/pub/MAPS/test_data/.

Yes.  The Arima-MAPS tool requires a configuration file, a ChIP-Seq peak file generated from the same cell type and antibodies, and a genomic features file. If no HiChIP reference peaks are available from your ChIP experiments, ENCODE sources multiple alternative ChIP peaks for various chromatin proteins.
The files needed to run Arima-MAPS are available at https://github.com/ijuric/MAPS/tree/master/Arima_Genomics/.

A test dataset to verify the MAPS installation and configuration is available at ftp://ftp-arimagenomics.sdsc.edu/pub/MAPS/test_data/.
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