Analysis of chromatin interactions mediated by specific architectural proteins in Drosophila cells

Abstract

Chromosome conformation capture assays have been established, modified, and enhanced for over a decade with the purpose of studying nuclear organization. A recently published method uses in situ Hi-C followed by chromatin immunoprecipitation (HiChIP) to enrich the overall yield of significant genome-wide interactions mediated by a specific protein. Here we applied a modified version of the HiChIP protocol to retrieve the significant contacts mediated by architectural protein CP190 in D. melanogaster cells.

1. Introduction

Genome-wide architectural landscapes can be generated using Hi-C [1]; this approach shares template creation with its chromosome conformation capture assay predecessors. The major difference is the use of biotinylated nucleotides to fill in the overhangs of restriction fragments, which are then ligated and pulled down to retrieve all the interactions across the genome. The ligation step is performed after nuclear lysis under diluted conditions, and this has been shown to abate the number of meaningful ligation junctions [2]. A variant of the regular Hi-C method is in situ Hi-C, which has the advantage of reducing the number of false contacts arising from ligation proximity [3]. Several variations of the Hi-C technology have been developed to map interacting regions associated with a protein of interest with high resolution. These techniques include Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) [4] and, more recently, HiChIP [5]. HiChIP combines in situ Hi-C (crosslinking, digestion, ligation and sonication) and chromatin immunoprecipitation with antibodies against a protein of interest to enrich for interactions mediated by this protein. Here we have adapted the HiChIP method to characterize interactions mediated by the architectural protein CP190 in D. melanogaster Kc167 cells.

The protocol consists of fixation of DNA-DNA-protein interactions, lysis and digestion of the DNA, biotinylation and proximity ligation of the restriction fragments followed by DNA shearing and ChIP. Afterwards, the crosslinks between DNA and proteins are reversed, DNA is precipitated with ethanol and the ligation junctions containing biotin are pulled-down with streptavidin beads. Once the DNA is on the streptavidin beads, the ends of sheared DNA are repaired and biotin is removed from unligated ends. Final steps involve the preparation of an Illumina sequencing library with adaptors for paired-end sequencing and size selection to sequence the DNA of interest. Subsequent computational analysis and visualization of the data was done with Juicer and Juicebox [6,7]. Significant DNA looping interactions were detected using Capture Hi-C Analysis of Genomic Organization (CHiCAGO) [8]. CHiCAGO is a tool developed to erase bias associated to Capture Hi-C experiments. However, both Capture Hi-C and HiChIP rely on the enrichment of interactions linked to specific regions of the genome. Therefore, the data from these two techniques share similar properties, making CHiCAGO a suitable tool for HiChIP data analysis. CHiCAGO performs larger numbers of tests at regions with smaller number of expected interactions and a background correction with a model that accounts for expected interactions and sequencing artifacts to detect significant interactions [8]. Specific software to account for biases in HiChIP data is not yet available, therefore tools developed for the analysis of ChIA-PET data, such as Mango [14] or MICC [15], may be just as effective as CHiCAGO at identifying significant interactions. A comparison of these algorithms and the development of new computational methods are essential for the advancement of the field.

2. Materials

Deionized water is the solvent for all solutions unless otherwise noted. Prepare and store all reagents at room temperature unless indicated otherwise.

2.1. Crosslinking

1. D. melanogaster Kc167 cells [10]

2. SFX-Insect cell culture media (HyClone)

3. Formaldehyde (37%). Store in dark conditions.

4. 2.5 M glycine solution.

2.2. Lysis and restriction digest

1. Hi-C lysis buffer: 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 (NP-40). Add 500 μL of 1 M Tris-HCl pH 8.0 stock solution, 100 μL of 5 M NaCl stock solution and 500 μL of 20% Igepal CA-630 (NP-40) stock solution in a 50 mL tube. Add water to a volume of 50 mL. Store at 4°C.

2. 25X Protease Inhibitor stock. Store at 4°C.

3. 0.5% SDS solution.

4. 10% Triton X-100 Stock solution.

5. 10X DpnII Reaction Buffer

6. 1X DpnII restriction enzyme (10,000 U/mL)

2.3. End-repair and biotinylation

1. Fill-in master mix: Add per tube 67.5 μL of water, 45 μL of 1 mM biotin-16-dCTP, 4.5 μL of 10 mM dTTP, 4.5 μL of 10 mM dATP, 4.5 μL of 10 mM dGTP and 24 μL of 5 U/μL DNA polymerase I Large (Klenow) fragment.

2. Ligation Master mix: Add per tube 2007 μL of water, 360 μL of 10x NEB T4 DNA Ligase buffer, 300 μL of 10% Triton X-100, 18 μL of 20 mg/mL BSA and 15 μL 400 U/μL T4 DNA Ligase.

2.4. Bead preparation

1. Magnetic beads with recombinant Protein A and Protein G

2. Blocking buffer: 0.5% BSA/PBS. Store at 4°C.

3. Pre-immune rabbit serum or immunoglobulin

4. Anti-CP190 antibody

5. IP Dilution Buffer: 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA pH 8.0, 16.7 mM Tris-HCl pH 8.0, 16.7 mM NaCl. Add 50 μL of 10% SDS stock solution, 5.5 mL of 10% Triton X-100 stock solution, 120 μL of 0.5 M EDTA pH 8.0 stock solution, 835 μL of 1 M Tris-HCl pH 8.0 stock solution and 1.67 μL of 1 M NaCl stock solution in a 50 mL tube. Add water to a volume of 50 mL. Store at 4°C.

2.5. DNA shearing

1. Nuclei Lysis Buffer: 50 mM Tris-HCl pH 8, 10 mM EDTA pH 8.0, 1% SDS. Add 2.5 mL of 1 M Tris-HCl pH 8.0 stock solution, 1 mL of 0.5 M EDTA pH 8.0 stock solution and 5 mL of 10% SDS stock solution in a 50 mL tube. Add water to a volume of 50 mL. Store at 4°C.

2. 25X Protease Inhibitor stock. Store at 4°C.

3. RNase A 10 mg/mL aliquot. Store at −20°C.

4. Proteinase K (20 mg/mL). Store at −20°C.

5. Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0. Use only in the laminar flow hood. Store at 4°C.

6. Glycogen aliquot. Store at −20°C.

7. 3 M sodium acetate solution.

8. 100% ethanol solution. Store at 4 °C.

9. 70% ethanol solution. Store at 4 °C.

10. 2% Agarose Gel. Weigh 1 g of Agarose for 50 mL of 1X TAE buffer, dissolve until solution is translucent for 3–5 min in microwave.

11. 1 X TAE Buffer: 40 mM Tris, 20 mM acetic acid, 1 mM EDTA pH 8.0.

12. 6X Gel loading dye, no SDS

13. 100 bp DNA ladder

14. Sonicator

2.6. Chromatin Immunoprecipitation

1. IP dilution buffer (previously described in 2.4 Bead preparation section)

2. IP Wash Buffer Low salt: 0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris-HCl pH 8.0, 150 mM NaCl. Add 500 μL of 10% SDS stock solution, 5 mL of 10% Triton X-100 stock solution, 200 μL of 0.5 M EDTA pH 8.0 stock solution, 1 mL of 1 M Tris-HCl pH 8.0 stock solution and 1.5 mL of 5 M NaCl stock solution in a 50 mL tube. Add water to a volume of 50 mL.

3. IP Wash Buffer High salt: 0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8, 20 mM, Tris-HCl pH 8.0, 500 mM NaCl. Add 500 μL of 10% SDS stock solution, 5 mL of 10% Triton X-100 stock solution, 200 μL of 0.5 M EDTA pH 8.0 stock solution, 1 mL of 1 M Tris-HCl pH 8.0 stock solution and 5 mL of 5 M NaCl stock solution in a 50 mL tube. Add water to a volume of 50 mL.

4. LiCl Buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.25 M LiCl, 1% Igepal CA-630 (NP-40), 1% DOC. Add 500 μL of 1 M Tris-HCl pH 8.0 stock solution, 100 μL of 0.5 M EDTA pH 8.0 stock solution, 3.125 mL of 4 M LiCl stock solution, 2.5 mL of 20% Igepal CA-630 (NP-40) and 5 mL of 10% DOC stock solution in a 50 mL tube. Add water to a volume of 50 mL.

5. IP Elution Buffer: 0.1 M NaHCO₃, 1% SDS. Prepare at the time of use. Add 100 μL of 1 M NaHCO₃, and 100 μL of 10% SDS. Add water to a total volume of 1.5 mL.

6. 5 M NaCl solution.

7. 0.5 M EDTA pH 8.0 stock solution

8. Elution Buffer: 10 mM Tris-Cl, pH 8.5

9. Proteinase K (20 mg/mL). Store at −20°C.

2.7. Biotin/Streptavidin pull-down and Preparation for Sequencing

1. Low-binding 1.5 mL tubes

2. Magnetic beads with recombinant streptavidin. Store at 4°C.

3. Tween Wash Buffer (TWB): 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA pH 8.0, 1 M NaCL, 0.05% Tween 20. Add 250 μL of 1 M Tris-HCl pH 7.5 stock solution, 50 μL of 0.5 M EDTA pH 8.0 stock solution, 10 mL of 5 M NaCl stock solution, 250 μL of 10% Tween 20 stock solution in a 50 mL tube. Add water to a volume of 50 mL.

4. Binding buffer (2X): 10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 2 M NaCl. Add 150 μL of 1 M Tris-HCl pH 7.5 stock solution, 30 μL of 0.5 M EDTA pH 8.0 stock solution, 6 mL of 5 M NaCl stock solution in a 15 mL tube. Add water to a volume of 15 mL.

2.8. Preparation of Illumina sequencing libraries

1. End-repair Master Mix: Add per tube 25 nM dNTPmix, 10 U/μL T4 Polynucleotide Kinase, 3 U/μL T4 DNA polymerase I, 5 U/μL DNA polymerase I large (Klenow) fragment

2. 1X NEB buffer 2. Store at −20°C

3. 10 nM dATP aliquot. Store at −20°C

4. Klenow Fragment (3´→ 5´ exo-) DNA Polymerase I (5,000 U/mL). Store at −20°C

5. 1X Quick ligase reaction buffer: Original stock is 2X concentration, dilute to reach 1X concentration.

6. Quick ligase enzyme

7. 2X KAPA SYBR FAST qPCR Kit aliquot. Store at −20°C

8. Refer to [11] for sequence information on adaptors and PCR primers.

9. Agencourt AMPure XP beads. Store at −20°C

10. 80% ethanol solution. Must be made before usage.

3. Methods

3.1. Crosslinking

1. Kc167 cells were grown to 80% confluence in SFX medium at 25°C. Use ~100 ×10⁶ as total number of cells.

2. In a 15 mL tube, pellet cells at 600 xg for 10 min at room temperature (22–25°C). Discard supernatant.

3. Add 10 mL of SFX medium and pipette to resuspend cells.

4. Divide into two 15 mL tubes, 5 mL in each one.

5. Add formaldehyde to a final concentration of 1% (see Note 1).

6. Incubate 7 min at room temperature on a rocker.

7. Add glycine to a final concentration of 0.2 M.

8. Incubate 5 min at room temperature on a rocker.

9. Pellet cells at 300 xg for 5 min at 4°C.

10. Discard supernatant into an appropriate collection container (see Note 2).

3.2. Lysis and restriction digest

1. dd 1.5 mL of ice cold Hi-C lysis buffer. Mix by pipetting and move to a 2 mL tube.

2. Add 60 μL of 25x Protease Inhibitor (PI).

3. Incubate on ice 20 minutes.

4. Pellet cells at 2500 xg for 5 min at 4°C and discard supernatant.

5. Add 1.5 mL of ice cold Hi-C lysis buffer. Mix by pipetting.

6. Pellet cells at 2500 xg for 5 min at 4°C and discard supernatant.

7. Add 150 μL of 0.5% SDS and pipette to resuspend cells.

8. Incubate 5 min at 65°C.

9. Add 435 μL of water and 75 μL of 10% Triton X-100.

10. Incubate 15 min at 37°C.

11. Add 75 μL of 10x DpnII buffer and 300 U of DpnII.

12. Digest overnight at 37°C.

3.3. End repair and biotinylation

1. Incubate digest reaction at 65°C for 10 min to inactivate DpnII.

2. Cool to room temperature.

3. Add 150 μL of the Fill-in Master Mix.

4. Mix by pipetting and incubate at 37°C for 45 min.

5. Pass the sample to a 15 mL tube.

6. Add 2700 μL of Ligation Master Mix to the sample.

7. Mix by inverting and incubate 4 hr at room temperature with gentle rocking.

3.4. Bead preparation

Prepare Pre-clear and Antibody beads during the 1.5 hr biotinylation incubation.

• 1.Pre-Clear beads: Add 10 μL of protein A beads and 10 μL of protein G beads in a 2 mL clean tube.

Antibody beads: Add 20 μL of protein A beads and 20 μL of protein G beads in a 2 mL clean tube.

• 2.Wash step:
• 2.1Add 1 mL of Blocking Buffer to each tube.
• 2.2Rotate for 5 min at room temperature.
• 2.3Collect on magnet stand.
• 2.4Remove supernatant.
• 3.Repeat wash step 2 more times.
• 4.Add 500 μL of Blocking Buffer to each tube.
• 5.Pre-Clear beads: Add 10 μL of pre-immune rabbit serum (or IgG).

Antibody beads: The amount of antibody depends on its efficiency.

• 6.Incubate the beads at 4°C on rotator for at least 4 hr (see Note 3).
• 7.Collect on magnet stand and remove supernatant.
• 8.Wash with Blocking Buffer:
• 8.1Add 1 mL of Blocking Buffer to each tube.
• 8.2Rotate for 2 min at room temperature.
• 8.3Collect on magnet stand.
• 8.4Remove supernatant.
• 9.Wash with IP Dilution Buffer.
• 9.1Add 1 mL of ice cold IP Dilution Buffer.
• 9.2Rotate for 2 min at room temperature.
• 9.3Collect on magnet stand.
• 9.4Remove supernatant.
• 10.Repeat wash with IP Dilution Buffer.
• 11.Add 50 μL of ice cold IP Dilution Buffer.

Beads are ready for steps in ChIP section.

3.5. DNA shearing

• 1.Pellet nuclei from ligation reaction at 1503 xg for 8 min at 4°C and discard supernatant.
• 2.Add 1.5 mL of ice cold Nuclei Lysis Buffer. Mix by pipetting and move to a 15 mL tube.
• 3.Add 60 μL of 25x PI.
• 4.Incubate in ice for 15 min.
• 5.Shear DNA to obtain DNA fragments between 250–350 bp (see Note 4 and 5).
• 6.Pellet cell debris at 21130 xg for 10 min at 4°C.
• 7.Transfer supernatant (chromatin) from 15 mL tube to a 1.5 mL clean tube.
• 8.Verify sonication quality (see Note 6):
• 8.1Add 15 μL of post-shear supernatant in a 2 mL clean tube.
• 8.2Add 185 μL of IP Dilution Buffer
• 8.3Add 0.5 μL of 10 mg/mL RNaseA
• 8.4Reverse cross link by heating to 95°C for 5 min.
• 8.5Add 4 μL of 10 mg/mL proteinase K.
• 8.6Incubate at 50°C for 2 hr.
• 8.7Extract DNA with Phenol:Chloroform:Isoamyl alcohol (P:C:I, 25:24:1)
• 8.7.1Add 1 volume of P:C:I (~205 μL) and vortex well.
• 8.7.2Spin at 21130 xg for 5 min at 4°C.
• 8.7.3Transfer aqueous (top) layer to a 2 mL clean tube.
• 8.8Add 1 μL of 20 mg/mL glycogen.
• 8.9Add 1/10 volume of 3 M Sodium Acetate.
• 8.10Add 2.5 volume of ice cold 100% Ethanol.
• 8.11Incubate 30 min at −80°C.
• 8.12Pellet DNA at 21130 xg for 10 min at 4°C and discard supernatant.
• 8.13Add 1 mL of 70% Ethanol.
• 8.14Pellet DNA at 21130 xg for 10 min at 4°C and discard supernatant.
• 8.15Let pellet air dry (5–10 min).
• 8.16Add 10 μL of water and resuspend the pellet.
• 8.17Run on a 2% agarose gel. Target is 250–300 bp.

3.6. Chromatin Immunoprecipitation

• 1.Add the sample from step 7 in DNA Shearing section to pre-clear beads.
• 2.Incubate 2 hr at 4°C with rotation.
• 3.Take 75 μL out to use as “input” reference. Can store at 4°C overnight.
• 4.Place the remaining solution on a magnet stand.
• 5.Move supernatant to tube with antibody coated beads.
• 6.Incubate at 4°C overnight with rotation.
• 7.Place on magnet stand and remove supernatant.
• 8.Add 1 mL of Low Salt Wash Buffer with PI freshly added. Mix by pipetting and move onto a 1.5 mL tube.
• 9.Incubate 5 min at room temperature with rotation.
• 10.Washes: Place on magnet stand and remove supernatant; add 1 mL of Wash Buffer with PI to a final concentration of 1x; mix by inverting.
• 10.1Wash with Low Salt Wash Buffer 2 more times.
• 10.2Wash with High Salt Wash Buffer 2 times.
• 10.3Wash with LiCl Buffer 2 times.
• 10.4Wash with TE buffer 1 time.
• 11.Place on magnet stand and remove supernatant.
• 12.Elution step:
• 12.1Add 400 μL of fresh IP elution buffer.
• 12.2Vortex for 2 seconds.
• 12.3Incubate 15 min at 68 °C and vortex 2 seconds each 2 minutes.
• 12.4Centrifuge at 21130 xg for 1 min at room temperature.
• 12.5Place on magnet stand.
• 13.Move supernatant to a clean tube.
• 14.Add 20 μL of 5 M NaCl, 8 μL of 0.5 M EDTA pH 8.0 and 16 μL of 1 M Tris-HCl pH 8.0.
• 15.Incubate for 6 hr at 68°C to reverse cross-link.
• 16.Add 8 μL of proteinase K.
• 17.Incubate at 50°C for 1 hr.
• 18.Cool to room temperature.
• 19.Extract DNA with Phenol:Chloroform:Isoamyl alcohol (P:C:I, 25:24:1)
• 19.1Add 400 μL volume of P:C:I and vortex well for one minute.
• 19.2Spin at 21130 xg for 5 min at 4°C.
• 19.3Transfer aqueous (top) layer to a 1.5 mL clean tube.
• 20.Precipitate the DNA:
• 20.1Add 1 μL of 20 mg/mL glycogen.
• 20.2Add 40 μL volume of 3 M Sodium Acetate.
• 20.3Add 1 mL of ice cold 100% Ethanol.
• 20.4Incubate 20 min at −80°C. Can go overnight.
• 22.Pellet DNA at 21130 xg for 20 min at 4°C and discard supernatant.
• 23.Add 1 mL of 70% ethanol.
• 24.Pellet DNA at 21130 g for 5 minutes at 4°C and discard supernatant.
• 25.Let pellet air dry (5–10 min).
• 26.Add 21 μL of Elution Buffer and resuspend the pellet.
• 27.Incubate at 37°C for 5 min to make sure pellet is properly dissolved.
• 28.Quantify the amount of DNA on a spectrophotometer.
• 29.Bring volume up to 100 μL with Elution Buffer.

3.7. Biotin/Streptavidin pull-down and Preparation for Sequencing

Perform remaining steps in 1.5 mL low-binding tubes

• 1.Add 400 μL of Tween Wash Buffer (TWB) to a clean tube.
• 2.Add 20 μL of MyOne Streptavidin T1 beads for every 5 μg of DNA.
• 3.Mix by inverting.
• 4.Separate on magnet stand and discard supernatant.
• 5.Add 400 μL of TWB.
• 6.Mix by inverting.
• 7.Separate on magnet stand and discard supernatant.
• 8.Add 100 μL of 2x Binding Buffer to resuspend the beads and add to the sample from step 29 in section 3.6 Chromatin Immunoprecipitation.
• 9.Incubate at room temperature for 20 min with rotation.
• 10.Separate on magnet stand and discard supernatant.
• 11.Wash with TWB:
• 11.1Add 600 μL of TWB
• 11.2Mix by inverting.
• 11.3Separate on magnet stand and discard supernatant.
• 12.Repeat wash with TWB one more time.

3.8. Preparation of Illumina sequencing libraries

• 1.Add 100 μL of 1x T4 ligase buffer and resuspend beads by pipetting. (see Note 5)
• 2.Separate on magnet stand and discard supernatant.
• 3.Resuspend beads in 100 μL of End-repair Master Mix.
• 4.Pipette gently to mix.
• 5.Incubate 30 min at room temperature with rotation.
• 6.Spin. Separate on magnet stand and discard supernatant.
• 7.Wash beads two times with TWB as before.
• 8.Add 100 μL of 1x NEB Buffer 2 and resuspend beads.
• 9.Separate on magnet stand and discard supernatant.
• 10.Add 100 μL of dATP Master Mix and resuspend by pipetting.
• 11.Incubate for 30 min at 37°C with rotation.
• 12.Spin. Separate on magnet stand and discard supernatant.
• 13.Wash beads two times with TWB as before.
• 14.Add 100 μL of 1x Quick ligase buffer and resuspend beads.
• 15.Separate on magnet stand and discard supernatant.
• 16.Add 50 μL of 1x Quick Ligase Buffer and resuspend beads.
• 17.Add 2 μL of Quick Ligase and 2 μL of the Illumina indexed adapter at 10 mM.
• 18.Incubate at room temperature for 15 min with rotation.
• 19.Spin. Separate on magnet stand and discard supernatant.
• 20.Wash beads two times with TWB as before.
• 21.Add 100 μL of Elution Buffer and resuspend beads.
• 22.Separate on magnet stand and discard supernatant.
• 23.Add 50 μL of Elution Buffer and resuspend beads.
• 24.Separate on magnet stand and discard supernatant.
• 25.Add 23 μL of Elution Buffer and resuspend beads. Amplify the library directly on the beads
• 26.Transfer the beads to a PCR tube.
• 27.Add 25 μL of Kapa SYBR FAST qPCR Kit Master Mix (2x), 2 μL of Illumina primers.
• 28.PCR parameters:
• 28.1Initial Denaturation 3 min at 98°C.
• 28.2PCR parameters: Denaturation 15 sec at 98°C; Annealing 30 sec at 60°C; Extension 45 sec at 72°C.
• 29.Run a total of 5 PCR cycles on a thermocycler.
• 30.Separate on a magnet stand and move reaction (supernatant) to a clean real-time PCR (qPCR) tube.
• 30.1Add more SYBR Green to a final concentration of 1x
• 31.qPCR parameters:
• 31.1Initial Denaturation 30 sec at 98°C.
• 31.2Cycle parameters: Denaturation 15 sec at 98°C; Annealing 30 sec at 60°C; Extension 45 sec at 72°C.
• 32.Run 4–6 cycles while monitoring on qPCR (see Note 8).
• 33.Move liquid from qPCR tube to a 1.5 mL clean tube.
• 34.Add 1.1x volumes of AMPure XP beads.
• 35.Incubate 5 min at room temperature on a rocker.
• 36.Separate on magnet stand and discard supernatant.
• 37.Without removing from magnetic stand, add 200 μL of 80% ethanol.
• 38.Wait for 30 seconds and discard ethanol.
• 39.Repeat last two steps.
• 40.Let pellet air dry (5–10 minutes).
• 41.Add 15 μL of Elution Buffer and resuspend the DNA.
• 42.Incubate 1 min at room temperature.
• 43.Separate on magnet stand and move the supernatant to a clean tube.
• 44.Quantify the amount of DNA on a spectrophotometer.
• 45.Send the library to sequencing by service provider for quality assessment and high throughput sequencing.

3.9. Data analysis

Most of the pipelines were ran through the terminal in a Linux-based system.

• 1.Set up
• 1.1Install Juicer software from http://aidenlab.org/juicer/.
• 1.2Install Juicebox software from http://aidenlab.org/juicebox/.
• 1.3Install R, download source file from https://cran.r-project.org/mirrors.html and choose the closest mirror to your location.
• 1.4Download CHiCAGO from https://bitbucket.org/chicagoTeam/chicago/downloads.
• 2.Read mapping and read-pair level filtering: Juicer (see Note 9)
• 2.1Create a directory called “fastq” and move sequence fastq files to this directory.
• 2.3Change the name of the files for each of the two mates obtained from paired-end sequencing. Each mate should have the name of the sample followed by either “R1” or “R2” to distinguish between the two mates. For example, sample_R1.fastq and sample_R2.fastq) (see Note 10).
• 2.4Go to the directory that contains the Juicer program “juicer.sh”
• 2.5
  Set the following parameters for Juicer and hit enter (see Note 11).

  • g genome: e.g. dm6
  • d path for “fastq” directory of the sample: e.g. /home/user/exp/
  • s restriction enzyme used: e.g. DpnII
  • p path for chromosome sizes file. This file should have two columns, the first is the name of the chromosome and the second one the length of the chromosome.
  • y enter path for restriction site file (locations of restriction sites in genome). This contains each chromosome and the positions of the cutting sites by the previously designated restriction enzyme in a row separated by a space, the last position of each row should be the end of that chromosome.
  • z enter path for sequence of the reference genome file in fasta format; the BWA index file must be in the same directory.

We use the following command:

$ juicer.sh -g dm6 -d /home/user/exp/ -s DpnII -p /home/user/exp/chromosome-sizes.txt -y /home/user/exp/restriction-sites.txt -z /home/user/exp/reference-sequence

• 2.6To verify the success of the alignment, consult that the “aligned” directory from the Juicer output contains the “inter.hic” file (see Note 12).
• 3.Normalization to account for experimental bias and extracting significant contacts: Juicebox and CHiCAGO
• 3.1
  Use the “juicebox dump” tool to retrieve a contact map. Set the following parameters and hit enter. (see Note 13 and 14)

  • Type of normalization: NONE (see Note 15).
  • Path of the .hic file: e.g. sample/aligned/inter.hic
  • Name of the first chromosome: either 2L, 2R, 3L, 3R, 4, X, Y.
  • Name of the second chromosome: either 2L, 2R, 3L, 3R, 4, X, Y.
  • Resolution desired: e.g. BP 5000 (see Note 16)
  • Name of output file: e.g. sample_firstchr_secondchr_obst.txt
• 3.2Create design files for CHiCAGO using dumped files from Juicer (see Note 17).
• 3.2.1Create a directory called “designDir”
• 3.2.2Create the .rmap file. This is a BED file with the positions of the bin with the columns chr, start, end, and fragmentID (see Note 18 and 19).
• 3.2.3Create the .baitmap file. This is also a BED file containing the bin with a bait, defined as the protein that is immunoprecipitated. It contains 5 columns: chr, start, end, fragmentID, baitAnnotation (see Note 20).
• 3.2.4
  Run Chicago/chicagoTools/makeDesignFiles.py to create .nbpb .poe and .npb files. Set the following parameters and hit enter (see Note 21).

  • designDir= path to the “designDir” directory
  • rmapfile= path to the .rmap file
  • baitmapfile=path to the .baitmap file
• 3.3Create .chinput file based on the juicebox dumped files. This 5-column file contains the contact frequency. The format is: baitID, otherendID, N, otherEndLen and distSign; where otherendID is the ID of the fragment or bin that is in the interacting pair of the bait, N is the number of reads detected for ligation products between the “bait” and “other end”, otherEndLen is the length of the “other-end” bin and distSign is the linear distance between the bait and other-end fragments, respectively [9] (see Note 22).
• 3.4Run CHiCAGO following the commands in http://regulatorygenomicsgroup.org/wp-content/uploads/Chicago_vignette.html
• 4.Visualization: Juicebox
• 4.1Raw data (see Note 23).
• 4.1.1The input file for visualization in Juicebox is inter.hic located inside the “aligned” directory.
• 4.1.2Open Juicebox
• 4.1.3Click on File>Open>Local and then choose the .hic file.
• 4.2CHiCAGO’s significant interactions: There are two ways to visualize CHiCAGO interactions. To visualize the data as a .hic matrix in juicebox go to 4.23, For an annotation track go to 4.27.
• 4.2.1CHiCAGO’s output in the designated directory contains the following directories: “data”, “diag_plots”, “enrichment_data examples”
• 4.2.2Enter “data” directory and find CP190_CHICAGO_washU_text.txt.
• 4.2.3Create .hic file using “juicebox pre” tool from CP190_CHICAGO_washU_text.txt file. to create a proper “juicebox pre” input file. It should be an 11-column file with the following format: readname, str1, chr1, pos1, frag1, str2, chr2, pos2, frag2, mapq1, mapq2; where str can be set to 0 for forward, anything else for reverse [7] (see Note 24) (e.g. 1 0 2L 73000 74000 0 2L 84000 85000 50 50)
• 4.2.4
  Set the parameters of “juicebox pre” tool and hit enter (see Note 25).

  • Pairwise Interaction BED file: CP190_CHICAGO_washU_text_5.txt
  • Output name: cp190_chicago.hic
  • Chromosome size file: path for chrom.sizes file. This file should have two columns, the first one is the name of the chromosome and the second one is the length of the chromosome.
  • Restriction map of D.melanogaster genome: enter path for restriction site file (locations of restriction sites in genome). This contains each chromosome and its restriction positions by the previously designated enzyme in a row separated by a space.
• 4.2.5Open Juicebox
• 4.2.6Click on File>Open>Local and then choose the CP190_chicago.hic file.
• 4.2.7Modify CP190_CHICAGO_washU_text.txt file to create a proper 2D annotation input file. It should be a 4-column file with the following format: readname, chr1, pos1, pos2, chr2, pos3, pos4, score (e.g. 2L 73000 74000 2L 2569000 2570000 35.6913535863312)
• 4.2.8Open Juicebox
• 4.2.9Open .hic file of the raw data. Click on File>Open>Local and then choose the CP190_raw.hic file.
• 4.2.10Load the significant interactions called by CHiCAGO using the modified version of the CP190_CHICAGO_washU_text.txt file. Click on Annotations>Load Basic Annotations>Dataset-specific 2D-features>Add 2D and select local file.

Figure 1.

Significant interactions associated with CP190 retrieved by CHiCAGO.

The upper and the lower triangle represent Hi-C and HiChIP raw data respectively with a 5 kb bin resolution. The black dots depict significant interactions obtained from CHiCAGO. The axes show the annotated genes for D. melanogaster and CP190 ChIP-seq peaks in this region. Figure was created using Juicebox v1.5.