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.
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. ~50,000 human cells or less, depending on the application).
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.
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 12µg of DNA. For most human cell lines, this usually corresponds to 3,000,000 to 4,000,000 cells as input.
Arima-HiChIP is compatible with cultured or primary mammalian cells.
ARIMA-HIC WORKFLOW
Each Arima Hi-C+ 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-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.
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. 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.
Arima currently provides pre-validated custom library prep user guides for KAPA HyperPrep, Swift Accel-NGS 2S Plus, Agilent SureSelect XT, Illumina TruSeq, NEBNext Ultra II, and others. Please contact an Arima representative to inquire about a pre-validated user guide for your preferred library prep kit.
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.
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.
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 plus and for a QC sequencing reaction. In order to generate sufficient material to use for hybridization for Capture 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 SureSelect XTHS protocol.
We at Arima will gladly help you with the bioinformatics to generate probes for your Arima capture experiment through our collaborator Agilent. The probe design with Arima is different for capture Hi-C. This is 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 SureSelectXTHS a minimum of 500ng of indexed input DNA is needed. The Arima-HiC indexed libraries can be used directly in the Agilent SureSelect XTHS protocol. We can also help you with designing custom Agilent probe sets for your Arima Capture- HiC experiment. For a custom Arima Capture-HiC probeset design you would simply need to share the genome coordinates and we will design a probeset for you taking into account the Arima cutsites that would be available for your review then purchase via Agilent.
Arima recommends the use of 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.
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.
The Arima-HiChIP protocol involves a 2 day workflow and 1 day for library preparation.
Arima recommends the use of pre-validated custom library prep user guides Swift Accel-NGS 2S Plus.
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 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.
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 personalized 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.
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 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, 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.
For a mammalian genome of 3Gb we recommend sequencing two biological replicates per biological condition. For high resolution analysis of A/B compartments, 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.
Per 1MB locus we recommend generating up to 1,000,000 on-target HiC contacts, which depending on the quality of the library will require approximately 10 million reads. Please contact technical support for more information.
We support the Chicago tools found at http://functionalgenecontrol.group/chicago. For the generation of HiC contact maps and identification and annotation of DNA loops and topological domains, Arima provides support for the use of open source software Juicerand recommends Juicebox for data visualization purposes.
Baitmap (and fragment files) for mouse and human are available for download. Please contact customer support for details.
We at Arima will gladly help you generate the baitmap file for your Arima Capture
-HiC experiment. It 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.
Arima provides 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 QC we recommend at least 500k paired end reads.
For Analysis of Arima HiChIP data we recommend the Arima-MAPS tool (https://github.com/ijuric/MAPS). Benchmarking with MAPS, FitHiChIP and HICCUPS and CriSPRi validated genomics loops revealed that MAPS has the highest sensitivity with a moderate false positive rate while minimizing computational time.
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 is sourcing multiple alternative ChIP peaks for various chromatin proteins.
The files needed to run MAPS for Arima 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/
