TITLE: HiChIP Protocol using Arima-HiC+ Kits
AUTHORS: Anthony Schmitt, Jon Belton, Sara Tin, Jonathan Kirkland, Derek Reid, Xiang Zhou

[DESCRIPTION]
HiChIP Workflow Overview
HiChIP is an experimental workflow that captures the structure (three-dimensional conformation) of genomes associated with a protein of interest. 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, bound to an antibody recognizing a protein of interest, immunoprecipitated, and purified to yield fragmented proximally-ligated DNA that was once bound in vivo to the protein of interest. The proximally-ligated DNA is subjected to a custom library preparation protocol utilizing a pre-validated commercially- available library prep kit. A separate HiChIP Library Prep user guide is provided that contains a custom protocol for enriching the proximally-ligated DNA for biotin and then converting the immunoprecipitated biotin-enriched DNA to HiChIP libraries.

Sequencing and Data Analysis
HiChIP libraries are sequenced via Illumina‱ sequencers in “paired-end” mode. The resulting data is referred to as HiChIP data. It is strongly recommended to process and analyze HiChIP using the publicly available tools Feather and MAPS (GitHub Link; Juric, 2019). This recommendation resulted from  benchmarking analyses of computational tools for HiChIP data analysis and a collaboration to optimize the outputs of MAPS for maximum compatibility. However, other tools such as HiC-Pro (Servant, 2015) and FitHiChIP (Bhattacharyya, 2019) may also be used. All publicly available tools require usage modifications and custom input files that are specific to HiChIP data using Arima-HiC+ kits, so please carefully review. It is recommended to leverage the WashU Epigenome Browser (http://epigenomegateway.wustl.edu).


[GUIDELINES]
Workflow Overview

 


[STEPS]
SECTION: Crosslinking – Cell Culture
1. Input: Cells collected from cell culture
Output: Crosslinked cells

Before you begin: This HiChIP workflow for mammalian cell lines begins with harvesting and crosslinking  12-15 million mammalian cells per biological replicate. Each reaction should only contain crosslinked cells comprising ~15µg of DNA (usually ~3-4 million mammalian cells), so this conservatively high quantity of cells recommended for crosslinking should be more than sufficient to complete the Estimating Input Amount and HiChIP Protocol. The excess cells account for less than expected DNA yields while also generating crosslinked cells for ChIP-seq experiments to define protein localization peaks. The crosslinking protocol below involves several cell pelleting centrifugations. For these centrifugations, pellet at the speed and duration you normally would for your specific cell type. Alternatively, we generally recommend centrifuging for 5 min at 1000 x G.
SECTION: Crosslinking – Cryopreserved Cells
2. Input: Cryopreserved cells
Output: Crosslinked cells

Before you begin: We recommend that the Arima-HiChIP workflow for mammalian cell lines begin with the crosslinking of cells harvested from cell culture, however, under certain circumstances one can also crosslink cells preserved in a cryogenic “freeze” media such as a mixture of complete cell culture media, FBS, and DMSO. A typical example would be cells that were once cultured and then collected at 5 million cells per mL in cryogenic “freeze” media, and stored in a liquid nitrogen tank. The crosslinking protocol below involves several cell pelleting centrifugations. During these centrifugations, pellet your specific cell types at a speed and duration as you normally would. Alternatively, we generally recommend centrifuging for 5 min at 500 x G.
SECTION: Crosslinking – Large Animal Tissue
3. Input: Fresh-frozen large animal tissue
Output: Crosslinked nuclei

Before you begin: The Arima-HiChIP workflow for large animal tissues begins with the pulverization and crosslinking of fresh-frozen large animal tissue. For most vertebrates and large invertebrates that comprise dense tissues, begin by weighing 200-600mg of fresh frozen tissue, and record this measured mass. The measured mass will be used later in this protocol and the following Estimating Input Amount – Large Animals protocol. For some applications, less than 200mg can be used, particularly when sample quantity is scarce. Note that this crosslinking protocol requires the handling of liquid nitrogen, dry ice, and severely cold equipment. Please use extra caution and wear cold-resistant gloves and appropriate PPE as needed.
SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4. Input: Crosslinked cells
Output: Purified genomic DNA

Before you begin: Arima-HiChIP reactions are optimally performed on crosslinked cells comprising 15µg of DNA. This amount of DNA ensures efficient Arima-HiC biochemistry, optimal chromatin immunoprecipitation performance, and sufficient Arima-HiChIP library complexity for the validated histone modification or transcription protein targets. The Estimating Input Amount protocol is required if one does not know how many crosslinked cells will comprise 15µg of DNA. The Estimating Input Amount protocol measures the amount of DNA obtained per 0.5 million crosslinked cells, in duplicate, and uses this to estimate the optimal cellular input for an Arima-HiChIP reaction. The Arima-HiC+ kit contains sufficient reagents to perform the Estimating Input Amount protocol on up to 8 samples, in duplicate, totaling up to 16 Estimating Input Amount reactions if needed. This protocol should be performed in microfuge tubes. This section concludes with a descriptive example of how to estimate the optimal number of crosslinked cells to use per Arima-HiChIP reaction.
SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.1. Thaw the two 0.5 million cell aliquots prepared during the Crosslinking protocol. 


 

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.2. Add 209.5 µL of a master mix containing the following reagents: 

 
 
 
SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.3. Add 20 µL of Buffer E, mix gently by pipetting, and incubate at 55 °C for 30 min.

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.4. Incubate at 68 °C for at least 180 min.

 

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.5. Incubate at Room temperature for 10 min. 


 

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.6. Add 225 µL of DNA Purification Beads, mix thoroughly, and incubate at Room temperature for 5 min.

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.7. Place sample against magnet, and incubate until solution is clear.
SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.8. Discard supernatant. While sample is still against magnet, add 500 µL of freshly prepared 80% ethanol, and incubate at Room temperature for 1 min.

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.9. Discard supernatant. While sample is still against magnet, add 500 µL of 80% ethanol, and incubate at Room temperature for 1 min.

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.10. Discard supernatant. While sample is still against magnet, incubate beads at Room temperature for 3-5 min. to air-dry the beads.

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.11. Remove sample from magnet, resuspend beads thoroughly in 50 µL of Elution Buffer, and incubate at Room temperature for 5 min.

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.12. Place sample against magnet, incubate until solution is clear, and transfer supernatant to a new tube.
SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.13. Quantify sample using a Qubit following the manufactueres instructions. Use 2 µL of DNA for the Qubit assay. The total DNA yield corresponds to the amount of DNA obtained from 0.5 million mammalian cells.

SECTION: Estimating Input Amount – Mammalian Cell Culture and Cryopreserved Cells
4.14. Estimate how many mammalian cells to use per Arima-HiChIP reaction. See the example below: 

Example: We recommend aiming to use crosslinked cells comprising 15 µg of DNA per Arima-HiChIP reaction. If an average of 2 µg of DNA was obtained per 0.5 million mammalian cells as calculated in Step 13, one can estimate that 3.75 million crosslinked cells should be used per Arima-HiChIP reaction (15 µg of DNA). Additionally, please note that the crosslinked cell pellet for one Arima-HiChIP reaction should occupy no more than 20 µL of volume in the sample tube. If the crosslinked cell pellet comprises 15 µg of DNA but occupies greater than 20 µL of volume, aliquot the cells into multiple Arima-HiChIP reactions such that the sum of the DNA input from all reactions is 15 µg and each cell pellet occupies no more than 20 µL of volume, or contact Technical Support for additional guidance.

SECTION: Estimating Input Amount – Large Animal Protocol
5. Input: Pulverized Crosslinked Tissue
Output: Purified genomic DNA

Before you begin: Arima-HiChIP reactions are optimally performed on crosslinked cells comprising 15 µg of DNA. This amount of DNA ensures efficient Arima-HiC biochemistry, optimal chromatin immunoprecipitation performance, and sufficient Arima-HiChIP library complexity for the validated histone modification or transcription protein targets. The Estimating Input Amount protocol is required if one does not know how many crosslinked cells will comprise 15 µg of DNA. The Estimating Input Amount – Large Animal protocol measures the amount of DNA obtained per 10 mg of unprocessed tissue, in duplicate, and uses this to estimate the optimal input amount for an Arima-HiChIP reaction. The Arima-HiC+ kit contains sufficient reagents to perform the Estimating Input Amount protocol on up to 8 samples, in duplicate, totaling up to 16 Estimating Input Amount reactions if needed. This protocol should be performed in microfuge tubes. This section concludes with a descriptive example of how to estimate the optimal amount of input material to use per Arima-HiChIP reaction.

SECTION: Estimating Input Amount – Large Animal Protocol
5.1. Thaw the two 10 mg DI Aliquots prepared during the Crosslinking – Large Animal protocol. 


 

SECTION: Estimating Input Amount – Large Animal Protocol
5.2. Add 209.5 µL of a master mix containing the following reagents:

 
 
 
SECTION: Estimating Input Amount – Large Animal Protocol
5.3. Add 20 µL of Buffer E, mix gently by pipetting, and incubate at 55 °C for 30 min

SECTION: Estimating Input Amount – Large Animal Protocol
5.4. Incubate at 68 °C for at least 180 min.

 


SECTION: Estimating Input Amount – Large Animal Protocol
5.5. Incubate at Room temperature for 10 min. 


 

SECTION: Estimating Input Amount – Large Animal Protocol
5.6. Add 225 µL of DNA Purification Beads, mix thoroughly, and incubate at Room temperature for 5 min.

SECTION: Estimating Input Amount – Large Animal Protocol
5.7. Place sample against magnet, and incubate until solution is clear.
SECTION: Estimating Input Amount – Large Animal Protocol
5.8. Discard supernatant. While sample is still against magnet, add 500 µL of freshly prepared 80% ethanol, and incubate at Room temperature for 1 min.

SECTION: Estimating Input Amount – Large Animal Protocol
5.9. Discard supernatant. While sample is still against magnet, add 500 µL of 80% ethanol, and incubate at Room temperature for 1 min.

SECTION: Estimating Input Amount – Large Animal Protocol
5.10. Discard supernatant. While sample is still against magnet, incubate beads at Room temperature for 3-5 minutes to air-dry the beads.

SECTION: Estimating Input Amount – Large Animal Protocol
5.11. Remove sample from magnet, resuspend beads thoroughly in 50 µL of Elution Buffer, and incubate at Room temperature for 5 min.

SECTION: Estimating Input Amount – Large Animal Protocol
5.12. Place sample against magnet, incubate until solution is clear, and transfer supernatant to a new tube.
SECTION: Estimating Input Amount – Large Animal Protocol
5.13. Quantify sample using a Qubit following manufacturers protocol. Use 2 µL of DNA for the Qubit assay. The total DNA yield corresponds to the amount of DNA obtained from 0.5 million mammalian cells.

SECTION: Estimating Input Amount – Large Animal Protocol
5.14. Estimate how many mammalian cells to use per Arima-HiChIP reaction. See the example below: 

Example: We recommend aiming to use crosslinked material comprising 15 µg of DNA per Arima-HiChIP reaction. If an average of 2 µg of DNA was obtained from 10 mg of original input material as calculated in Step 13 then the sample yields 200 ng of DNA per mg of input tissue. This value is highly variable between different tissue types do to the density of cells in the tissue and the how difficult the tissue is to dissociate. To achieve an input of  , for this example, 75 mg of the original tissue mass must be input into each reaction. If 200 mg of tissue total were processed during the dissociation then 0.375x of the processed material must be added to one reaction. Additionally, please note that the crosslinked cell pellet for one Arima-HiChIP reaction should occupy no more than 20 µL of volume in the sample tube. If the crosslinked cell pellet comprises 15 µg of DNA but occupies greater than 20 µL of volume, aliquot the cells into multiple Arima-HiChIP reactions such that the sum of the DNA input from all reactions is 15 µg and each cell pellet occupies no more than 20 µL of volume, or contact Technical Support for additional guidance.

SECTION: HiChIP Protocol
6. Input: Crosslinked cells containing 15 µg of DNA
Output: Immunoprecipitated proximally-ligated DNA fragments

Overview: Arima-HiChIP begins with generating biotin-labelled proximally-ligated chromatin using Arima-HiC chemistry (Sections 1-3). The proximally-ligated chromatin is then sheared using Covaris (Section 8) or Diagenode Bioruptor (Section 9) instruments and bound to an antibody overnight. In parallel, Protein A beads are blocked overnight (Section 3.3) to prepare for immunoprecipitation. On the following day, the antibody-bound proximally-ligated chromatin is immunoprecipitated on Protein A beads, reverse crosslinked, and purified (Section 3.4). The resulting proximally-ligated DNA may be subject to quality control analysis described in the Quality Control section, and is then converted to sequence-ready Arima-HiChIP libraries following a custom protocol in a separate Arima-HiChIP Library Preparation user guide.
SECTION: HiChIP Protocol
6.1. Perform Arima-HiC

Before you begin: The pellet of previously crosslinked cells or nuclei for one Arima-HiChIP reaction should occupy no more than 20 µL of volume and should be devoid of any residual liquid. If the cell pellet occupies greater than 20 µL of volume, aliquot the cells such that the sum of the DNA input from all reactions is between 10-15 µg and each cell pellet occupies no more than 20 µL of volume, or contact Technical Support for additional guidance. Note that Steps 2 – 3 require consecutive heated incubations. Make sure your thermal device(s) are pre-set to 62 °C and 37 °C for these incubations to avoid prolonged waiting periods before and between heated incubations.


 

SECTION: HiChIP Protocol
6.2. Aliquot cells for the Arima-HiChIP reactions by resuspending the crosslinked cell pellet from above in a total volume of 1 mL of  . Visually measure the volume of the cell pellet (50-100 µL) and subtract it from 1ml to determine the amount of cold 1x PBS to resuspend the cells in. For instance, if the cell pellet is 50 µL of volume, resuspend it in 950 µL of cold  .

SECTION: HiChIP Protocol
6.3. Aliquot the amount of cells required for 15 µg based on Estimating Input Amount (Sections 4-5) above by taking the proportion of the cell pellet that corresponds to the amount of cells determined to yield 15 µg. For instance, if the cell pellet has 10 million cells and the Estimating Input Amount calculated that 4 million cells is needed for each HiChIP reaction then remove 400 µL from the resuspended cell pellet and place in a new 1.5 mL. 
 


SECTION: HiChIP Protocol
6.30. Discard supernatant. Once completed, store sample on on ice and immediately proceed to the appropriate Chromatin Shearing and Antibody Binding sections depending on use of a Covaris (Section 7) or Diagenode Bioruptor Pico (Section 8) instrument.

SECTION: HiChIP Protocol
6.29. Pellet sample by centrifugation at 10.000 x g, 10 min, 4 °C.

SECTION: HiChIP Protocol
6.28. If sample is in a PCR tube or PCR plate, transfer sample to a clean 1.5 mL.

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6.27. Mix gently by inversion, and then immediately transfer 10 µL of sample into a new tube labelled “Ligation QC”. Store the Ligation QC sample at -20 °C until later use in a following Quality Control section, and proceed to the next step with the remaining sample.

SECTION: HiChIP Protocol
6.26. If, the user intends to optimize shearing settings for specific samples, follow the instructions in Appendix F before proceeding with step 26. If shearing will be performed with the recommended settings provided by Arima, or with settings previously optimized for the sample type/sonicator combination being used then please proceed with step 26.
SECTION: HiChIP Protocol
6.25. If shearing settings are to be optimized using the instructions in Appendix F, it is recommended to store test samples and proceed with shearing optimization on the following day.
SECTION: HiChIP Protocol
6.24. Store samples at -20 °C for up to 3 nights before proceeding with Chromatin Shearing. Sample should be stored as-is. There is no need to remove supernatant at this time. After sample is removed from -20 °C storage, proceed with step 24.

SECTION: HiChIP Protocol
6.23. Mix gently by pipetting, and incubate at Room temperature for 15 min.

SECTION: HiChIP Protocol
6.22. Add 82 µL of a master mix containing the following reagents: 

 
 
 
 
 
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6.21. Mix gently by pipetting, and incubate at Room temperature for 45 min.

SECTION: HiChIP Protocol
6.20. Add 16 µL of a master mix containing the following reagents:

 
 
 
SECTION: HiChIP Protocol
6.19. Transfer sample to a clean 1.5 mL, PCR tube, or PCR plate.

SECTION: HiChIP Protocol
6.18. Resuspended sample pellet in 75 µL Deionized Water by gently pipetting 5-10 times.

SECTION: HiChIP Protocol
6.17. Carefully discard supernatant, without disturbing the sample pellet.
SECTION: HiChIP Protocol
6.16. Pellet sample by centrifugation at 10.000 x g, 10 min, 4 °C.

SECTION: HiChIP Protocol
6.15. Gently add 1.5 mL Deionized Water, without disturbing the sample pellet.

SECTION: HiChIP Protocol
6.14. Carefully discard supernatant, without disturbing the sample pellet.
SECTION: HiChIP Protocol
6.13. Pellet sample by centrifugation at 10.000 x g, 10 min, 4 °C.

SECTION: HiChIP Protocol
6.12. Transfer sample to a clean 1.5 mL.

SECTION: HiChIP Protocol
6.11. Mix gently by pipetting, and incubate at 37 °C for 60 min. If using a thermal cycler, set the lid temperature to 85 °C.

SECTION: HiChIP Protocol
6.10. Add 12 µL of a master mix containing the following reagents: 

 
 

 

SECTION: HiChIP Protocol
6.9. Add 20 µL of Stop Solution 2, mix gently by pipetting, and incubate at 37 °C for 15 min. If using a thermal cycler, set the lid temperature to 85 °C.

SECTION: HiChIP Protocol
6.8. Add 24 µL of Conditioning Solution, mix gently by pipetting, and incubate at 62 °C for 10 min. If using a thermal cycler, set the lid temperature to 85 °C.

SECTION: HiChIP Protocol
6.7. Resuspend the aliquot of crosslinked cells prepared in step 3 above in 20 µL of Lysis Buffer in a tube or a well of a PCR plate, and incubate at 4 °C for 20 min.

SECTION: HiChIP Protocol
6.6. Re-freeze the crosslinked cell pellet on dry ice or liquid nitrogen, and store at -80 °C.

SECTION: HiChIP Protocol
6.5. Remove the supernatant from the cell pellets.
SECTION: HiChIP Protocol
6.4. Pellet the cell aliquot and the remainder of the resuspended crosslinked cell pellet by centrifugation at 1.000 x g, 5 min, 4 °C.

SECTION: HiChIP Protocol
7. Chromatin Shearing and Antibody Binding (Covaris Workflow)

Before you begin: We have extensively validated the Covaris S220 instrument for chromatin shearing within the Arima-HiChIP Protocol, using the Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm tubes (Covaris SKU: 520045). Other Covaris instruments have also been used successfully. Please do not attempt to substitute any reagents or change any steps as this may result in a decrease in chromatin shearing efficiency and reproducibility.
SECTION: HiChIP Protocol
7.1. Add 130 µL of cold CS Buffer to sample pellet, and resuspend by gentle pipette mixing.

SECTION: HiChIP Protocol
7.2. Transfer 130 µL of sample to a Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm tube

SECTION: HiChIP Protocol
7.3. Shear each sample using the Covaris instrument. Always store samples on on ice except when the sample is being sheared.

SECTION: HiChIP Protocol
7.4. If shearing was optimized using the instructions in Appendix F, apply the optimized settings determined for each specific sample type.
SECTION: HiChIP Protocol
7.5. If recommended shearing settings are to be used, exemplary shearing parameters using a Covaris S220 instrument on 2% crosslinked cells are provided below. If using a different Covaris instrument, ensure that the total energy delivered is the same as the below recommendations. Please contact Technical Support for any questions regarding chromatin shearing or for additional guidance.

 
 


SECTION: HiChIP Protocol
7.6. Transfer sheared sample to a 1.5 mL tube. Gently flick or spin the Covaris microTUBE to ensure all sample has been collected and transferred. 


 

SECTION: HiChIP Protocol
7.7. Transfer 10 µL of sheared sample into a new tube labelled “Shearing QC”. Store the Shearing QC sample at -20 °C until later use in the Quality Control section, and proceed to the next step with the remaining sample.

SECTION: HiChIP Protocol
7.8. Preclear the chromatin by adding 30 µL of Protein A Beads to a new 1.5 mL  for each sample.

SECTION: HiChIP Protocol
7.9. Place Protein A Beads against magnet, and incubate until solution is clear.
SECTION: HiChIP Protocol
7.10. Discard supernatant.
SECTION: HiChIP Protocol
7.11. Remove Protein A Beads from magnet, resuspend Protein A Beads in 880 µL of cold MR1 Buffer, and mix gently by pipetting. Do not vortex.

SECTION: HiChIP Protocol
7.12. Add the remaining 120 µL of sheared sample from step 7.6, to bring the total volume to 1 mL. Mix gently by pipetting. Do not vortex.

SECTION: HiChIP Protocol
7.13. Incubate for >1hr at 4 °C on a nutator, rotator, orbital shaker or equivalent device.

SECTION: HiChIP Protocol
7.14. Sample should rotate at 4 °C while completing Reverse Crosslinking and DNA Purification in section 9 of the protocol. 
 

SECTION: HiChIP Protocol
8. Chromatin Shearing and Antibody Binding – Diagenode Bioruptor Pico Workflow

Before you begin: We have validated the Diagenode Bioruptor Pico instrument for chromatin shearing within the Arima-HiChIP Protocol. Note that the efficiency of chromatin shearing on the Diagenode Bioruptor Pico is very sensitive to the sample shearing volume. The total sample volume after the completion of Step 8.3 should be exactly as indicated.
SECTION: HiChIP Protocol
8.1. Add 110 µL of cold R1 Buffer to sample pellet, and resuspend by gentle pipette mixing.

SECTION: HiChIP Protocol
8.2. Incubate at 4 °C for 20 min.

SECTION: HiChIP Protocol
8.3. Transfer exactly 110 µL of sample to a Diagenode 0.65 mL.

SECTION: HiChIP Protocol
8.4. Shear sample using the Diagenode Bioruptor Pico instrument

 

 

SECTION: HiChIP Protocol
8.5. Transfer sheared sample to a 1.5 mL. Gently flick or spin the Diagenode 0.65 mL to ensure all sample has been collected and transferred. 


 

SECTION: HiChIP Protocol
8.6. Transfer 10 µL of sheared sample into a new tube labelled “Shearing QC”. Store the Shearing QC sample at -20 °C until use in the Quality Control section, and proceed to the next step with the remaining sample.

SECTION: HiChIP Protocol
8.7. Preclear the chromatin by adding 30 µL of Protein A Beads to a new 1.5 mL for each sample.

SECTION: HiChIP Protocol
8.8. Place Protein A Beads against magnet, and incubate until solution is clear.
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8.9. Discard supernatant.
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8.10. Remove Protein A Beads from magnet, resuspend Protein A Beads in 900 µL of cold R1 Buffer, and mix gently by pipetting. Do not vortex.

SECTION: HiChIP Protocol
8.11. Add to the remaining 100 µL of sheared sample from step 8.5 above, to bring the total volume to 1 mL. Mix gently by pipetting. Do not vortex.

SECTION: HiChIP Protocol
8.12. Incubate for >1 hr at 4 °C on a nutator, rotator, orbital shaker or equivalent device.

SECTION: HiChIP Protocol
8.13. Sample should rotate at 4 °C while completing the Reverse Crosslinking and DNA Purification in section 9 of the protocol. 
 


SECTION: HiChIP Protocol
9. Reverse Crosslinking and DNA Purification

Overview: In this section, chromatin from the Ligation QC and Shearing QC aliquots collected during the Arima-HiChIP Protocol is reverse crosslinked and purified (Aliquots were collected in steps 7.6 and 8.5). The Shearing yield will be used to adjust the amount of Antibody added during the Immunoprecipitation to ensure high specificity capture of epitopes in the immunoprecipitation.

Before you begin: This sub-section describes the reverse crosslinking and purification of DNA from the Ligation QC and Shearing QC aliquots collected during the Arima-HiChIP Protocol. The DNA purified during this sub-section will be used in sub-sections 4.1 and 4.2 that follow. This protocol can be performed in microfuge tubes, PCR tubes, or PCR plates.


 

SECTION: HiChIP Protocol
9.1. Thaw all Ligation QC and Shearing QC aliquots.
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9.2. Add 90 µL Elution Buffer to each Ligation and Shearing QC aliquot, to bring the total volume to 100 µL. 


 

SECTION: HiChIP Protocol
9.3. Add 20.3 µL of a master mix containing the following reagents: 

 
 
 
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9.4. Add 11.4 µL of Buffer E, mix gently by pipetting, and incubate as follows. If using a thermal cycler, set the lid temperature to 85 °C.

 
 

 

 


SECTION: HiChIP Protocol
9.5. Add 120 µL of DNA Purification Beads, mix thoroughly, and incubate at Room temperature for 5 min.

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9.6. Place sample against magnet, and incubate until solution is clear.
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9.7. Discard supernatant. While sample is still against magnet, add 200 µL of freshly prepared 80% ethanol, and incubate at Room temperature for 1 min.

SECTION: HiChIP Protocol
9.8. Discard supernatant. While sample is still against magnet, add 200 µL of 80% ethanol, and incubate at Room temperature for 1 min.

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9.9. Discard supernatant. While sample is still against magnet, incubate beads at Room temperature for 3-5 min. to air-dry the beads.

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9.10. Remove sample from magnet, resuspend beads thoroughly in 30 µL of Elution Buffer, and incubate at Room temperature for 5 min.

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9.11. Place sample against magnet, incubate until solution is clear, and transfer supernatant to a new tube.
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9.12. Quantify sample using a Qubit. Use 2 µL of DNA for the Qubit assay.

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9.13. Record the sample concentration of the Shearing QC samples in the Arima-HiChIP QC Worksheet on the ChIP Efficiency tab under “Shearing QC Samples”. Use the worksheet to calculate the ChIP Efficiency value. Store remaining sample at -20 °C until use in the Chromatin Fragmentation QC section below (Section 4.2). 


 

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10. Antibody Addition

Overview: The success of an Arima HiChIP library is highly dependent on the success of the antibody binding step. This can vary depending on the type of antibody and available genome target locations. Too much antibody will bind non-specific targets and result in poor library specificity. To little antibody can result in poor library complexity. The steps below provide guidance for how to calculate the optimal mass of antibody to add to each sample to ensure the best chance of a successful HiChIP reaction.

Before you begin: This sub-section describes how to calculate the optimal mass of antibody to add to an individual reaction based on shearing yield, which is needed before proceeding with the Antibody binding step. The calculation is based on the following formula:

Mass of Antibody = Shearing Yield x Antibody Ratio
SECTION: HiChIP Protocol
10.1. Remove pre-clearing samples from 4 °C (step 7.14 or 8.13), and place against magnet, and incubate until solution is clear.

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10.2. Transfer the supernatant (precleared chromatin) to a new 1.5 mL. 
 


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10.3. Use the worksheet in the Arima-HiChIP QC Worksheet on the ChIP Efficiency tab under “Shearing QC Samples”
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10.4. Add antibody as calculated in column H, “ug of Antibody to add to sample”, and mix the entire sample gently by pipetting. Do not vortex.
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10.5. The ratio for each antibody is listed in the Table below. This is calculated automatically in the Arima HiChIP QC Worksheet on the ChIP Efficiency tab under “Shearing QC Samples”, column H, by multiplying the g of shearing yield by the ratio listed in the table on the next page. 
 

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10.6. Incubate at 4 °C overnight on a nutator, rotator, orbital shaker or equivalent device.

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10.7. Immediately after setting up the overnight incubation in the prior step, proceed to the Bead Blocking sub-section (Section 11) directly below.
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11. Bead Blocking 

Before you begin: This sub-section prepares the Protein A beads that will be used in the Chromatin Immunoprecipitation sub-section (Section 12) on the following day. To increase the specificity of the chromatin immunoprecipitation reaction, the Protein A beads are “blocked” overnight via incubation with R2 Buffer, which contains BSA.
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11.5. Incubate at 4 °C overnight on a nutator, rotator, orbital shaker or equivalent device.

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11.4. Remove Protein A Beads from magnet, resuspend Protein A Beads in 500 µL R2 Buffer, and mix gently by pipetting. Do not vortex.

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11.3. Discard supernatant.
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11.2. Place Protein A Beads against magnet, and incubate until solution is clear.
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11.1. Add 30 µL of Protein A Beads to a new 1.5 mL for each sample.

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12. Chromatin Immunoprecipitation (ChIP)

Before you begin: The chromatin immunoprecipitation sub-section comprises conjugating the antibody-bound chromatin to blocked Protein A Beads, rigorous washing, reverse crosslinking and purification of the immunoprecipitated proximally-ligated DNA fragments. This section requires use of a thermomixer pre-cooled to 4 °C, which may take at least 120 min. Alternatively, the thermomixer can be placed in a cold room or refrigerator.
 


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12.1. Set a thermomixer to 4 °C or place in a cold room or refrigerator and allow to cool for at least 120 min for the thermomixer to reach 4 °C.

SECTION: HiChIP Protocol
12.2. Remove blocked Protein A Beads from 4 °C incubation, place against magnet, and incubate until solution is clear.

SECTION: HiChIP Protocol
12.3. Discard supernatant.
SECTION: HiChIP Protocol
12.4. Remove Protein A Beads from magnet, add 1 mL antibody-bound chromatin to the blocked Protein A Beads, and mix by pipetting until homogeneous. 
SECTION: HiChIP Protocol
12.5. Incubate at 4 °C for 240 min on a nutator, rotator, orbital shaker or equivalent device.
 

SECTION: HiChIP Protocol
12.6. Place sample against magnet, and incubate until solution is clear.
SECTION: HiChIP Protocol
12.7. Discard supernatant. Resuspend sample in 1 mL R1 Buffer, mix thoroughly by pipetting, and incubate on a thermomixer at 4 °C for 3 min. with 1000 rpm shaking.

SECTION: HiChIP Protocol
12.8. Repeat Steps 6-7 two times using R1 Buffer, for a total of 3 R1 Buffer washes.
SECTION: HiChIP Protocol
12.9. Repeat Steps 6-7 two times using R3 Buffer.
SECTION: HiChIP Protocol
12.10. Repeat Steps 6-7 one time using LC Buffer.
SECTION: HiChIP Protocol
12.11. Repeat Steps 6-7 two times using LTE Buffer.
SECTION: HiChIP Protocol
12.12. Place sample against magnet, and incubate until solution is clear.
SECTION: HiChIP Protocol
12.13. Discard supernatant. 
 

SECTION: HiChIP Protocol
12.14. Resuspend sample in 174 µL Elution Buffer and transfer the resuspended sample to a LoBind 1.5 mL. The resuspended sample can also be transferred into a PCR tube or plate for the completion of Steps 12.15-12.16 below. 
 



SECTION: HiChIP Protocol
12.15. Add 35.5 µL of a master mix containing the following reagents: 

 

SECTION: HiChIP Protocol
12.16. 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. 
 
 
 
 
SECTION: HiChIP Protocol
12.17. If sample is in a PCR tube or PCR plate, transfer sample into a LoBind 1.5 mL.
 


SECTION: HiChIP Protocol
12.18. Add 230 µL of DNA Purification Beads, mix thoroughly, and incubate at Room temperature for 5 min.

SECTION: HiChIP Protocol
12.19. Place sample against magnet, and incubate until solution is clear.
SECTION: HiChIP Protocol
12.20. Discard supernatant. While sample is still against magnet, add 700 µL of freshly prepared 80% ethanol, and incubate at Room temperature for 1 min.

SECTION: HiChIP Protocol
12.21. Discard supernatant. While sample is still against magnet, add 700 µL of 80% ethanol, and incubate at Room temperature for 1 min.

SECTION: HiChIP Protocol
12.22. Discard supernatant. While sample is still against magnet, incubate beads at Room temperature for 3 – 5 min. to air-dry the beads.

SECTION: HiChIP Protocol
12.23. Remove sample from magnet, resuspend beads thoroughly in 50 µL of Elution Buffer, and incubate at Room temperature for 5 min.

SECTION: HiChIP Protocol
12.24. Place sample against magnet, incubate until solution is clear, and transfer supernatant to a new tube.
 



SECTION: HiChIP Protocol
12.25. Quantify sample using a Qubit. Use 2 µL of DNA for the Qubit assay.

SECTION: HiChIP Protocol
12.26. Record the sample concentration in the Arima-HiChIP QC Worksheet on the ChIP Efficiency tab under “ChIP QC Samples”.
SECTION: HiChIP Protocol
12.27. If >100ng of DNA is available, transfer 10 ng of sheared sample into a new tube labelled “ChIP QC” and store at -20 °C until later use in the Quality Control section.

SECTION: HiChIP Protocol
12.28. Store remaining sample at -20 °C until ready to proceed to library preparation following an accompanying Arima-HiChIP Library Preparation user guide using Swift Biosciences Accel-NGS 2S Plus DNA Library Kit (Cat # 21024 or 21096).

SECTION: Crosslinking – Cell Culture
1.1. Harvest cells from cell culture using standard protocols and pellet cells by centrifugation.
SECTION: Crosslinking – Cryopreserved Cells
2.1. Fill a 15 mL conical tube with 4 mL of  .

SECTION: Crosslinking – Cryopreserved Cells
2.2. Thaw the cryopreserved cells in a 37 °C water bath.

Note: In the following step, the entire contents of the cryopreserved cell sample (i.e. cells and the cryogenic media) are transferred into the conical tube containing  . Do not centrifuge the cells to try and remove the cryogenic freeze media. The following step also assumes the cells are preserved in 1 mL of cryogenic freeze media, and transferring the cells into the   will bring the total volume to 5 mL. If the cells are not frozen in 1 mL of cryogenic freeze media, adjust the volume of   so that the total sample volume after Step 3 will be 5 mL.

SECTION: Crosslinking – Cryopreserved Cells
2.3. Gently transfer cells, including the cryogenic freeze media, into the conical tube containing 4 mL of  , bringing the total volume to 5 mL.

SECTION: Crosslinking – Cryopreserved Cells
2.4. Add 286 µL of  , bringing the final formaldehyde concentration to 2%.

SECTION: Crosslinking – Cryopreserved Cells
2.5. Mix well by inverting 10 times and incubate at 10 Room temperature for 10 min 

SECTION: Crosslinking – Cryopreserved Cells
2.6. Add 460 µL of Stop Solution 1, mix by inverting 10 times and incubate at Room temperature10 min .

SECTION: Crosslinking – Cryopreserved Cells
2.7. Place sample at on ice  and incubate for 15 min.

SECTION: Crosslinking – Cryopreserved Cells
2.8. Pellet cells by centrifugation and discard supernatant.
SECTION: Crosslinking – Cryopreserved Cells
2.9. Resuspend cells in 1 mL  .

SECTION: Crosslinking – Cryopreserved Cells
2.10. Aliquot cells into several new 1.5 mL tubes, two with 0.5 million per aliquot, and one with the remaining 11-14 million. Mix sample by inversion between aliquots.

SECTION: Crosslinking – Cryopreserved Cells
2.11. Pellet cells in all aliquots by centrifugation.
SECTION: Crosslinking – Cryopreserved Cells
2.12. Discard supernatant, leaving only the crosslinked cell pellets and no residual liquid.
SECTION: Crosslinking – Cryopreserved Cells
2.13. Freeze samples on dry ice or liquid nitrogen, and store at -80 °C until ready to proceed to the Estimating Input Amount protocol the following section.

SECTION: Crosslinking – Large Animal Tissue
3.1. Prepare buffers TLB1, TLB2, SS using the recipes in Appendix A and cool on ice. Also, cool a centrifuge large enough to spin 50 mL  at 4 °C and cool 10 mL of   on ice per sample.

SECTION: Crosslinking – Large Animal Tissue
3.2. Embed a mortar, with a pestle inside of it, and a 50 mL  onto a cooler of dry ice and allow to cool. Cool a spatula by placing it in the 50 mL  for later use.

SECTION: Crosslinking – Large Animal Tissue
3.3. Pour liquid nitrogen into the mortar until the entire pestle tip is submerged. Allow liquid nitrogen to evaporate completely to cool the mortar and pestle.
SECTION: Crosslinking – Large Animal Tissue
3.4. Pour liquid nitrogen into the mortar until the entire pestle tip is submerged. Transfer 200-600 mg frozen large animal tissue into mortar containing liquid nitrogen. Allow liquid nitrogen to evaporate just enough for the tissue to stay submerged.

SECTION: Crosslinking – Large Animal Tissue
3.5. Pulverize the tissue in the mortar using the pestle until the sample resembles a fine powder like corn starch or powdered sugar. Ensure the tissue is always submerged in liquid nitrogen. Carefully re-fill the mortar with liquid nitrogen as necessary. The pulverization process should take at least 5 min per sample and some tissue types may take longer. The goal is to pulverize until the tissue resembles a fine powder without visible chunks. 

Note: Using cold-resistant gloves is highly recommended to handle the mortar and pestle.
SECTION: Crosslinking – Large Animal Tissue
3.6. Once the sample resembles a fine powder, allow liquid nitrogen in the mortar to evaporate just enough for sample to stay submerged in a viscous slurry.
SECTION: Crosslinking – Large Animal Tissue
3.7. Carefully pour pulverized tissue and remaining liquid nitrogen from the mortar into the 50 mL . Ensure the tube does not overflow with liquid nitrogen.

SECTION: Crosslinking – Large Animal Tissue
3.9. Submerge 50 mL into dry ice to keep all the pulverized animal tissue frozen.

Note: DO NOT cap the 50 mL until all the liquid nitrogen has completely evaporated.

SECTION: Crosslinking – Large Animal Tissue
3.10. Allow liquid nitrogen in 50 mL to evaporate completely. 

Note: If pulverizing multiple animal tissue samples in a single day, keep the pulverized sample on dry ice and repeat Steps 1-9 on the remaining samples using clean equipment and consumables until all samples have been pulverized. Then, proceed to the next step and complete the remaining protocol on all samples simultaneously.
SECTION: Crosslinking – Large Animal Tissue
3.11. Remove sample tube from dry ice.
SECTION: Crosslinking – Large Animal Tissue
3.12. Incubate sample tube for 10 min on ice.

SECTION: Crosslinking – Large Animal Tissue
3.13. Add 5 mL of cold   and resuspend via pipetting with a 1ml pipette.

SECTION: Crosslinking – Large Animal Tissue
3.14. Pellet sample by centrifugation at 1.000 x g at 4 °C for 5 min.

SECTION: Crosslinking – Large Animal Tissue
3.15. Discard supernatant.
SECTION: Crosslinking – Large Animal Tissue
3.16. Add 3 mL of TLB1 and resuspend via pipetting with a 1ml pipette.

SECTION: Crosslinking – Large Animal Tissue
3.8. Using the cooled spatula from Step 2, transfer any remaining pulverized large animal tissue from the mortar into the 50 mL.

SECTION: Crosslinking – Large Animal Tissue
3.17. Incubate sample tube for 20 min at 4 °C, occasionally mix by inverting. Proceed to steps 18 and 19 below during the incubation.

SECTION: Crosslinking – Large Animal Tissue
3.45. Snap freeze the aliquot cell pellets on dry ice for 5 minor liquid nitrogen for 1 min, then store at -80 °C for up to one year.

SECTION: Crosslinking – Large Animal Tissue
3.18. Prepare a fresh 50 mL with a 40µm cell strainer on top, per sample.

SECTION: Crosslinking – Large Animal Tissue
3.19. Rinse the 40µm cell strainer with 5-10 mL, discard flow through.

SECTION: Crosslinking – Large Animal Tissue
3.20. Add suspended sample to the cell strainer and allow the liquid to drip into the 50 mL. Stir gently with a clean spatula to assist flow through. Continue Stirring until most of the liquid has flowed through the filter.

SECTION: Crosslinking – Large Animal Tissue
3.21. Rinse cell strainer with 2 mL of TLB2, stir with spatula until most of the liquid flows through.


 


SECTION: Crosslinking – Large Animal Tissue
3.22. Tap the cell strainer on the top of the 50 mL to dislodge any remaining drips of liquid. Discard the cell strainer and cap the tube. Ground tissue that is less than 40 µm will be in the 50 mL. This will include small cell clumps, single cells and nuclei.

SECTION: Crosslinking – Large Animal Tissue
3.23. Pellet sample by centrifugation at 1.000 x g, 4 °C  for 5 min. Reduce the slowdown speed of the centrifuge so that the pellet will not be disturbed when the centrifuge stops.

SECTION: Crosslinking – Large Animal Tissue
3.24. Discard supernatant, making sure not to disturb the pellet.
SECTION: Crosslinking – Large Animal Tissue
3.25. Resuspend sample in 1 mL of TLB2, using a 1ml pipette.

SECTION: Crosslinking – Large Animal Tissue
3.26. Overlay 3 mL of buffer SS (Sucrose Solution) carefully using a 5ml serological pipette by pipetting no faster than 0.1ml per second. The SS should form a layer on top of the cell suspension.

SECTION: Crosslinking – Large Animal Tissue
3.27. Pellet sample by centrifugation at 2.500 x g, 5 min, 4 °C for 5 min. Reduce the slowdown speed of the centrifuge so that the sucrose gradient will not be disturbed when the centrifuge stops.

SECTION: Crosslinking – Large Animal Tissue
3.28. Discard supernatant.
SECTION: Crosslinking – Large Animal Tissue
3.29. Resuspend sample in 1 mL of cold  .

SECTION: Crosslinking – Large Animal Tissue
3.30. Pellet sample by centrifugation at 2.500 x g, 5 min, 4 °C for 5 min.

SECTION: Crosslinking – Large Animal Tissue
3.31. Discard supernatant.


 

SECTION: Crosslinking – Large Animal Tissue
3.32. Resuspend sample in 5 mL of room temperature  .

SECTION: Crosslinking – Large Animal Tissue
3.33. Add 286 µL of  , to bring the final formaldehyde concentration to 2%.

SECTION: Crosslinking – Large Animal Tissue
3.34. Mix well by inverting 10 times and incubate at Room temperature for 5 min . Mix the sample every 2-3 mins with gentle inversion.

SECTION: Crosslinking – Large Animal Tissue
3.35. Add 460 µL of Stop Solution 1, mix well by inverting 10 times and incubate at Room temperature for 5 min. Mix the sample every 2-3 mins with gentle inversion.

SECTION: Crosslinking – Large Animal Tissue
3.36. Place sample on ice and incubate for 15 min.

SECTION: Crosslinking – Large Animal Tissue
3.37. Pellet sample by centrifugation at 2.500 x g, 5 min, 4 °C.

SECTION: Crosslinking – Large Animal Tissue
3.38. Discard supernatant.
SECTION: Crosslinking – Large Animal Tissue
3.39. Resuspend sample in 3 mL  .

SECTION: Crosslinking – Large Animal Tissue
3.40. Pellet sample by centrifugation at 2.500 x g, 5 min, 4 °C.

SECTION: Crosslinking – Large Animal Tissue
3.41. Discard supernatant.
SECTION: Crosslinking – Large Animal Tissue
3.42. Resuspend sample in 1 mL  , and transfer to a 1.5 mL.

SECTION: Crosslinking – Large Animal Tissue
3.43. To prepare for the Estimating Input Amount – Large Animals protocol in a following section, mix the sample by pipetting and then immediately aliquot sample such that 2 aliquots (DI Aliquots) contains the equivalent of 10mg of the original pulverized large animal tissue, while the rest of the aliquots each contain the equivalent of ~20-25% (Storage Aliquots) of the pulverized large animal tissue. Mix sample by inversion between aliquots to ensure all aliquots are equally homogeneous. For example, if processing 200mg of pulverized large animal tissue, then take 5% of the suspended material. The remaining aliquots containing 20-25% are meant to be saved as sample material for the Arima-HiC Protocol.
SECTION: Crosslinking – Large Animal Tissue
3.44. Pellet all aliquots by centrifugation at 2.500 x g, 5 min, 4 °C. Discard supernatant leaving behind only the sample pellet and no residual liquid.

SECTION: Crosslinking – Cell Culture
1.2. Resuspend cells in cell culture media, obtain a cell count by hemocytometer or automated cell counting methods.
SECTION: Crosslinking – Cell Culture
1.3. Transfer 12-15 million to be crosslinked into a new 15 mL conical tube, pellet cells by centrifugation and remove supernatant.

SECTION: Crosslinking – Cell Culture
1.4. Resuspend cells in 5 mL of  Room temperature  .

Note: In the below step, add methanol-stabilized formaldehyde to crosslink cells at a final formaldehyde concentration of 2%. Please DO NOT use other formaldehyde concentrations. 
SECTION: Crosslinking – Cell Culture
1.5. Add 286 µL of  , to bring the final formaldehyde concentration to 2%. 

SECTION: Crosslinking – Cell Culture
1.6. Mix well by inverting 10 times and incubate at Room temperature for 10 min.

SECTION: Crosslinking – Cell Culture
1.7. Add 460 µL of Stop Solution 1, mix well by inverting 10 times and incubate at Room temperature for 5 min.

SECTION: Crosslinking – Cell Culture
1.9. Discard supernatant.    
SECTION: Crosslinking – Cell Culture
1.8. Pellet cells by centrifugation.
SECTION: Crosslinking – Cell Culture
1.10. Resuspend cells in 1 mL  . 

SECTION: Crosslinking – Cell Culture
1.11. Aliquot cells into several new 1.5 mL, two with 0.5 0.5 million per aliquot, and one with the remaining 11-14 million. Mix sample by inversion between aliquots.

SECTION: Crosslinking – Cell Culture
1.12. Pellet cells in all aliquots by centrifugation.
SECTION: Crosslinking – Cell Culture
1.13. Discard supernatant, leaving only the crosslinked cell pellets and no residual liquid. 
SECTION: Crosslinking – Cell Culture
1.14. Freeze samples on dry ice or liquid nitrogen, and store at -80 °C until ready to proceed to the Estimating Input Amount protocol the following section.          

SECTION: Quality Control
13. Overview: In this section, multiple quality control analyses
are performed to assess the efficiency of Arima-HiC, chromatin shearing, and chromatin immunoprecipitation. In sub-section 4.1, use the Arima-HiChIP QC Worksheet on the Arima-QC1 tab under “Ligation QC
Samples” to calculate how much volume from the "Ligation QC” aliquot to
use as input to the Arima-QC1 sub-section below (Section 4.2) and store
remaining sample at -20°C until use in the Chromatin Fragmentation QC section.  The DNA size from
the proximally-ligated and sheared chromatin is analyzed to determine the
efficiency of Arima-HiC and chromatin shearing. Also if available, the DNA size
of the immunoprecipitated DNA is analyzed to confirm the expected size range of
the immunoprecipitated DNA. Lastly in sub-section 4.2,
the fraction of proximally-ligated DNA that has been labelled with
biotin is analyzed using DNA purified from the Ligation QC aliquot. All these
QC data are recorded and analyzed in the accompanying Arima-HiChIP QC Worksheet.  
SECTION: Quality Control
14. Chromatin Fragmentation QC

Before you begin: In this section, gel electrophoresis analysis will be used to evaluate the DNA size in the proximally-ligated and sheared chromatin, which determines the efficiency of Arima-HiC and chromatin shearing. This section will use the Ligation QC and Shearing QC aliquots purified in the previous section. If the ChIP QC aliquot has been collected during the Arima-HiChIP Protocol, this section can also be used to confirm the expected size range of the immunoprecipitated DNA. If performing this section concurrently with the Chromatin Immunoprecipitation protocol (Section 3.4), the ChIP QC aliquot will not be available yet.
SECTION: Quality Control
14.1. Thaw the Ligation QC and Shearing QC aliquots. Thaw the ChIP QC aliquot if it has already been collected. 
SECTION: Quality Control
14.2. Analyze the DNA size of the proximally-ligated and sheared chromatin from the Ligation and Shearing QC samples, respectively, and the ChIP QC sample if one is available. Use gel electrophoresis systems such as a Bioanalyzer, TapeStation, or FlashGel. Exemplary results from the FlashGel system are below.
 

SECTION: Quality Control
15. Arima-QC1

Before you begin: The following protocol quantifies the fraction of proximally-ligated DNA that has been labelled with biotin, and is a quality control metric after completing the Arima-HiChIP Protocol but before proceeding to library preparation. The Arima-QC1 protocol below uses QC Beads to enrich an aliquot of purified proximally-ligated DNA purified from the Ligation QC aliquot, which is quantified using a Qubitâ fluorometer. Unlike standard Qubitâ readings which involve quantifying a transparent unobstructed DNA sample, the Arima-QC1 value is obtained by quantifying DNA that is still bound to the QC Beads. This protocol can be performed in either plates or tubes. Set your thermal device (thermal cycler or thermomixer) to hold at 55°C. After completing the Arima-QC1 protocol, use the provided Arima-HiChIP QC Worksheet to determine the Arima-QC1 values.
SECTION: Quality Control
15.1. If necessary, thaw the Ligation QC samples prepared during Step 14 of the Reverse Crosslinking and DNA Purification protocol (Section 4.1) and transfer 75 ng of sample into a new tube labelled “Arima-QC1”. 

SECTION: Quality Control
15.2. Add Elution Buffer 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.

SECTION: Quality Control
15.3. Add 50 µL of QC Beads, mix thoroughly by pipetting, and incubate at Room temperature for 15 min.

SECTION: Quality Control
15.4. Place sample against magnet, and incubate until solution is clear.
SECTION: Quality Control
15.5. Discard supernatant, and remove sample from magnet.
SECTION: Quality Control
15.6. Wash beads by resuspending in 200µL of Wash Buffer, and incubate at 55C for 2 min.
SECTION: Quality Control
15.7. Place sample against magnet, and incubate until solution is clear.
SECTION: Quality Control
15.8. Discard supernatant, and remove sample from magnet.
SECTION: Quality Control
15.9. Wash beads by resuspending in 200µL of Wash Buffer, and incubate at 55C for 2 min.
SECTION: Quality Control
15.10. Place sample against magnet, and incubate until solution is clear.
SECTION: Quality Control
15.11. Discard supernatant, and remove sample from magnet.
SECTION: Quality Control
15.12. Wash beads by resuspending in 100µL of Elution Buffer.
SECTION: Quality Control
15.13. Place sample against magnet, and incubate until solution is clear.
SECTION: Quality Control
15.14. Discard supernatant, and remove sample from magnet.
SECTION: Quality Control
15.15. Resuspend beads in 7µL of Elution Buffer. Proceed to next step with resuspended beads. 
 

SECTION: Quality Control
15.16. Quantify the total amount of bead-bound DNA using Qubit. Use 2µL of thoroughly mixed bead-bound DNA for the Qubit assay.
SECTION: Quality Control
15.17. Determine the Arima-QC1 value by following the Arima-HiChIP 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.
SECTION: Quality Control
16. 
SECTION: Appendix A - Tissue Dissociation User-Supplied Buffers
17. Tissue Lysis Buffer 1 (TLB1), for 8 samples

 

 

SECTION: Appendix A - Tissue Dissociation User-Supplied Buffers
18. Tissue Lysis Buffer 2 (TLB2), for 8 samples

 

 

SECTION: Appendix A - Tissue Dissociation User-Supplied Buffers
19. Sucrose Solution (SS), for 8 samples

 

 

SECTION: Appendix B – Day 1 Universal User-Supplied Buffers
20. R2 Buffer

 

 

SECTION: Appendix B – Day 1 Universal User-Supplied Buffers
21. 
SECTION: Appendix C – Day 1 Covaris® User-Supplied Buffers
22. CS Buffer

 

 



SECTION: Appendix C – Day 1 Covaris® User-Supplied Buffers
23. MR1 Buffer

 

 

SECTION: Appendix D – Day 1 Bioruptor User-Supplied Buffers
24. R1 Buffer

 

 

SECTION: Appendix E – Day 2 Universal User-Supplied Buffers
25. R1 Buffer

 

 

SECTION: Appendix E – Day 2 Universal User-Supplied Buffers
26. R3 Buffer

 

 

SECTION: Appendix E – Day 2 Universal User-Supplied Buffers
27. LC Buffer

 

 

SECTION: Appendix E – Day 2 Universal User-Supplied Buffers
28. LTE Buffer – The LTE Buffer is a low EDTA (0.1mM) TE Buffer that does not need to be prepared fresh directly before use in the Arima-HiChIP Protocol.

 

 

SECTION: Appendix F – Optional Shearing Titration
29. Chromatin Shearing Optimization- Covaris Workflow

Before you begin: The below steps are intended to be performed if one or more samples processed are intended for shearing optimization. Note: we recommend that shearing optimization be conducted for each sample type of interest and for each shearing platform being used.  Following the below steps, the sample will likely be over-sheared and should not be included in subsequent steps.
SECTION: Appendix F – Optional Shearing Titration
30.2. Add 90µL Elution Buffer to each shearing optimization aliquot, to bring the total volume to 100µL.

 

SECTION: Appendix F – Optional Shearing Titration
30.1. Thaw all shearing optimization aliquots. 
SECTION: Appendix F – Optional Shearing Titration
30. Reverse Crosslinking of Shearing Optimization Aliquots

Before you begin: This sub-section describes the reverse crosslinking and purification of DNA from the shearing optimization aliquots collected above. This protocol can be performed in microfuge tubes, PCR tubes, or PCR plates.

 

SECTION: Appendix F – Optional Shearing Titration
29.1. Add 130µL of cold CS Buffer to sample pellet of the shearing optimization samples, and resuspend by gentle pipette mixing.
SECTION: Appendix F – Optional Shearing Titration
29.2. Transfer 130µL of sample to a Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm tube
SECTION: Appendix F – Optional Shearing Titration
29.3. Shear the sample following the steps below using a Covaris S220 instrument on 2% crosslinked cells.  Always store samples on ice except when the sample is being sheared.


 

SECTION: Appendix F – Optional Shearing Titration
29.4. 1 minute:
a.    Begin by sonicating the shearing optimization sample using the shearing parameters provided below under “1 minute”
b.    After completion of the 1 minute sonication, transfer 10uL of the sheared sample to a new tube labeled “1 minute”
c.     Add 10uL of CS buffer to the Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm tube which contains the shearing optimization sample

SECTION: Appendix F – Optional Shearing Titration
29.5. 3 minutes:
a.    Sonicate the shearing optimization sample again using the shearing parameters provided below under “3 minutes”
b.    After completion of the 3 minute sonication, transfer 10uL of the sheared sample to a new tube labeled “3 minutes”
c.     Add 10uL of CS buffer to the Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm tube which contains the shearing optimization sample
SECTION: Appendix F – Optional Shearing Titration
29.6. 5 minutes:
a.    Sonicate the shearing optimization sample again using the shearing parameters provided below under “5 minutes”
b.    After completion of the 5 minute sonication, transfer 10uL of the sheared sample to a new tube labeled “5 minutes”
c.   Add 10uL of CS buffer to the Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm tube which contains the shearing optimization sample
SECTION: Appendix F – Optional Shearing Titration
29.7. 7 minutes:
a.    Sonicate the shearing optimization sample again using the shearing parameters provided below under “7 minutes”
b.    After completion of the 7 minute sonication, transfer 10uL of the sheared sample to a new tube labeled “7 minutes”
c.     Add 10uL of CS buffer to the CovarismicroTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm tube which contains the shearing optimization sample
SECTION: Appendix F – Optional Shearing Titration
29.8. 9 minutes:
a.    Sonicate the shearing optimization sample again using the shearing parameters provided below under “9 minutes”
b.    After completion of the 9 minute sonication, transfer 10uL of the sheared sample to a new tube labeled “9 minutes”                                                      
c.     Only 10uL is required for reverse crosslinking. The remaining 120uL can be discarded.
SECTION: Appendix F – Optional Shearing Titration
29.9. Please contact Technical Support for any questions regarding chromatin shearing or for additional guidance.
SECTION: Appendix F – Optional Shearing Titration
30.3. Add 20.3µL of a master mix containing the following reagent:

 

SECTION: Appendix F – Optional Shearing Titration
30.4. Add 11.4µL of Buffer E, mix gently by pipetting, and incubate as follows. If using a thermal cycler, set the lid temperature to 85°C.

 
*Do not incubate at 68°C for longer than 90 min. unless doing so using a thermal cycler or thermomixer with a heated lid.
** To provide flexibility, this incubation can also be held overnight at 4°C, in which case, the sample may turn slightly opaque or have precipitation. Warm sample to room temperature to re-dissolve the precipitate before proceeding to purification.

 

SECTION: Appendix F – Optional Shearing Titration
30.5. Add 120µL of DNA Purification Beads, mix thoroughly, and incubate at RT for 5 min.
SECTION: Appendix F – Optional Shearing Titration
30.6. Place sample against magnet, and incubate until solution is clear.
SECTION: Appendix F – Optional Shearing Titration
30.7. Discard supernatant. While sample is still against magnet, add 200µL of 80% ethanol, and incubate at RT for 1 min.
SECTION: Appendix F – Optional Shearing Titration
30.8. Discard supernatant. While sample is still against magnet, add 200µL of 80% ethanol, and incubate at RT for 1 min.
SECTION: Appendix F – Optional Shearing Titration
30.9. Discard supernatant. While sample is still against magnet, incubate beads at RT for 3 – 5 min. to air-dry the beads.
SECTION: Appendix F – Optional Shearing Titration
30.12. Analyze the DNA size of the sheared chromatin from the shearing optimization aliquots.  200-800bp.  Use gel electrophoresis systems such as a Bioanalyzer, TapeStation, or FlashGelTM. Exemplary results from the FlashGel system are below.
 

SECTION: Appendix F – Optional Shearing Titration
30.11. Place sample against magnet, incubate until solution is clear, and transfer supernatant to a new tube.
SECTION: Appendix F – Optional Shearing Titration
30.10. Remove sample from magnet, resuspend beads thoroughly in 30µL of Elution Buffer, and incubate at RT for 5 min.
SECTION: Appendix F – Optional Shearing Titration
31. Chromatin Shearing Optimization- Diagenode Bioruptor Pico Workflow

Before you begin: The below steps are intended to be performed if one or more samples processed are intended for shearing optimization. Note: we recommend that shearing optimization be conducted for each sample type of interest and for each shearing platform being used.  Following the below steps, the sample will likely be over-sheared and should not be included in subsequent steps.
SECTION: Appendix F – Optional Shearing Titration
31.1. Add 110µL of cold R1 Buffer to sample pellet, and resuspend by gentle pipette mixing.
SECTION: Appendix F – Optional Shearing Titration
31.2. Incubate at 4C for 20 min.
SECTION: Appendix F – Optional Shearing Titration
31.3. Transfer exactly 110µL of sample to a Diagenode 0.65mL Microtube.
SECTION: Appendix F – Optional Shearing Titration
31.4. 10 Cycles:
1.   Shear sample using the Diagenode Bioruptor Pico instrument using 30” ON / 30” OFF cycling conditions and 10 cycles total. 
2.   After completion of the 5 cycle sonication, transfer 10uL of the sheared sample to a new tube labeled “10 cycles”.
3.   Add 10uL of R1 buffer to the Diagenode 0.65mL Microtube which contains the shearing optimization sample.
4.   Vortex and quick spin the sample in a microfuge to re-incorporate sample material that has been ejected out of solution onto the upper tube wall to provide more uniform and consistent shearing.  
 

SECTION: Appendix F – Optional Shearing Titration
31.5. 15 Cycles:
1.   Shear sample using the Diagenode Bioruptor Pico instrument using 30” ON / 30” OFF cycling conditions and 5 cycles total. 
2.    After completion of the 5 cycle sonication, transfer 10uL of the sheared sample to a new tube labeled “15 cycles”.
3.    Add 10uL of R1 buffer to the Diagenode 0.65mL Microtube which contains the shearing optimization sample.
4.   Vortex and quick spin the sample in a microfuge to re-incorporate sample material that has been ejected out of solution onto the upper tube wall to provide more uniform and consistent shearing.   
 

SECTION: Appendix F – Optional Shearing Titration
31.6. 20 Cycles:
1.       Shear sample using the Diagenode Bioruptor Pico instrument using 30” ON / 30” OFF cycling conditions and 5 cycles total. 
2.    After completion of the 5 cycle sonication, transfer 10uL of the sheared sample to a new tube labeled “20 cycles”.
3.    Add 10uL of R1 buffer to the Diagenode 0.65mL Microtube which contains the shearing optimization sample.
4.   Vortex and quick spin the sample in a microfuge to re-incorporate sample material that has been ejected out of solution onto the upper tube wall to provide more uniform and consistent shearing.
 

SECTION: Appendix F – Optional Shearing Titration
31.7. 25 Cycles:
1.       Shear sample using the Diagenode Bioruptor Pico instrument using 30” ON / 30” OFF cycling conditions and 5 cycles total. 
2.    After completion of the 5 cycle sonication, transfer 10uL of the sheared sample to a new tube labeled “25 cycles”.
3.    Add 10uL of R1 buffer to the Diagenode 0.65mL Microtube which contains the shearing optimization sample Vortex and quick spin the sample in a microfuge to re-incorporate sample material that has been ejected out of solution onto the upper tube wall to provide more uniform and consistent shearing.
 

SECTION: Appendix F – Optional Shearing Titration
31.8. 30 Cycles:
1.   Shear sample using the Diagenode Bioruptor Pico instrument using 30” ON / 30” OFF cycling conditions and 5 cycles total. 
2.   After completion of the 5 cycle sonication, transfer 10uL of the sheared sample to a new tube labeled “30 cycles”
3.   Only 10uL is required for reverse crosslinking.  The remaining 120uL can be discarded.
SECTION: Appendix F – Optional Shearing Titration
32. Reverse Crosslinking of Shearing Optimization Aliquots
SECTION: Appendix F – Optional Shearing Titration
32.1. Thaw all shearing optimization aliquots.
SECTION: Appendix F – Optional Shearing Titration
32.2. Add 90µL Elution Buffer to each shearing optimization aliquot, to bring the total volume to 100µL. 
 

SECTION: Appendix F – Optional Shearing Titration
32.3. Add 20.3µL of a master mix containing the following reagents:

 

SECTION: Appendix F – Optional Shearing Titration
32.4. Add 11.4µL of Buffer E, mix gently by pipetting, and incubate as follows. If using a thermal cycler, set the lid temperature to 85°C.

 
*Do not incubate at 68°C for longer than 90 min. unless doing so using a thermal cycler or thermomixer with a heated lid.
** To provide flexibility, this incubation can also be held overnight at 4°C, in which case, the sample may turn slightly opaque or have precipitation. Warm sample to room temperature to re-dissolve the precipitate before proceeding to purification.
 
 
SECTION: Appendix F – Optional Shearing Titration
32.5. Add 120µL of DNA Purification Beads, mix thoroughly, and incubate at RT for 5 min.
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32.6. Place sample against magnet, and incubate until solution is clear.
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32.7. Discard supernatant. While sample is still against magnet, add 200µL of 80% ethanol, and incubate at RT for 1 min.
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32.8. Discard supernatant. While sample is still against magnet, add 200µL of 80% ethanol, and incubate at RT for 1 min.
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32.9. Discard supernatant. While sample is still against magnet, incubate beads at RT for 3 – 5 min. to air-dry the beads.
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32.10. Remove sample from magnet, resuspend beads thoroughly in 30µL of Elution Buffer, and incubate at RT for 5 min.
SECTION: Appendix F – Optional Shearing Titration
32.11. Place sample against magnet, incubate until solution is clear, and transfer supernatant to a new tube.
SECTION: Appendix F – Optional Shearing Titration
32.12. Analyze the DNA size of the sheared chromatin from the shearing optimization aliquots.  The ideal size for sheared chromatin is 200-800bp. Use gel electrophoresis systems such as a Bioanalyzer, TapeStation, or FlashGelTM. Exemplary results from the FlashGel system are below.