CiFi: 3C Library Preparation for PacBio HiFi Sequencing

• Always use wide-bore tips to minimize DNA shearing
• Ensure reagents are thawed and mixed as directed. 
• Avoid overdrying beads during cleanup steps.
• Maintain accurate temperature control during incubations.
• Crosslinked pellets must be free of residual liquid before starting the reaction.
• Use DNA LoBind tubes throughout to minimize sample loss and preserve long DNA fragments.

• Wash 5-10 million cells three times in chilled 1X phosphate buffered saline (PBS) in a 50 mL centrifuge tube.
• Centrifuge at 500xg for 5 minutes at 4°C between each wash.
• Resuspend cells in 10 mL room temperature 1X PBS with 1% formaldehyde [EMD Millipore cat no. 818708] by gently pipetting with a wide bore tip.
• Incubate at room temperature for 10 minutes.

• Add 527 µL of 2.5 M glycine (final 125 mM) in 10.5 mL.
• Incubate for 5 minutes at room temperature followed by 10 minutes on ice.
• Pellet the cross-linked cells by centrifugation at 500xg for 5 minutes at 4°C.
• Wash the cross-linked cells with 1x PBS, remove the supernatant, and snap-freeze the pellet in liquid nitrogen. Store at -80°C until you start the protocol.

• Resuspend the cell pellet in a mixture of 50 μL of protease inhibitor cocktail [Sigma Aldrich cat no. P8340] in 500 μL of cold permeabilization buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630).
• Place on ice for 15 minutes.
• Centrifuge at 500xg for 10 minutes at 4°C.
• Aspirate the supernatant and replace with 200 μL of chilled 1.5X digestion reaction buffer [NEB] compatible with the restriction enzyme used.
• Centrifuge again at 500xg for 10 minutes at 4°C, then aspirate and resuspend in 300 μL of chilled 1.5X digestion reaction buffer.

• Add 33.5 μL of 1% w/v SDS [Thermo Fisher Scientific cat no. 15553027] to each cell suspension.
• Incubate for exactly 10 minutes at 65°C with gentle agitation.
• Place on ice immediately afterwards.
• Quench the SDS by adding 37.5 μL of 10% v/v Triton X-100 [Sigma Aldrich cat no. 93443] for a final concentration of 1%.
• Incubate for 10 minutes on ice.

• Permeabilized cells are then digested with a final concentration of 1 U/μL of  DpnII [NEB], brought to volume with nuclease-free water to achieve a final 1X digestion reaction buffer in 450 μL.
• Mix by gentle inversion and incubate in a thermomixer at 37°C for 18 hours with periodic < 1000 rpm rotation (< 30 seconds every 15 minutes) to prevent condensation inside the lid

• DpnII restriction digests are heat-inactivated at 65°C for 20 minutes with 300 rpm rotation. Place on ice immediately afterwards. 

• Set up proximity ligation at room temperature with the following reagents:
• 100 μL of 10X T4 DNA ligase buffer [NEB]
• 10 μL of 10 mg/mL BSA
• 50 μL of T4 Ligase [NEB M0202L]
• Total volume of 1000 μL with nuclease-free water.
• Cool to 16°C and incubate for 6 hours with gentle rotation.

• Treat samples with:
• 100 μL 20 mg/mL Proteinase K [Thermo Fisher Scientific cat no. 25530049]
• 100 μL 10% SDS [Thermo Fisher Scientific cat no. 15553027]
• 500 μL 20% v/v Tween-20 [Sigma Aldrich cat no. P9416]
• Total volume of 2000 μL with nuclease-free water.
• Incubate in a thermomixer at 56°C for 18 hours with < 1000 rpm rotation (< 30 seconds every 15 minutes) to prevent condensation inside the lid.

1. Cool the sample on ice after completing the reverse cross-linking incubation (56 °C, ~18 hr).
2. Transfer the entire sample (~2.0–2.2 mL) to a 5 mL DNA LoBind centrifuge tube or a 15 mL centrifuge tube.
3. Rinse the original tube with 200 µL nuclease-free water and add this to the same 5 mL tube (final volume ~2.2 mL).
4. Add an equal volume of chilled phenol:chloroform:isoamyl alcohol (25:24:1)
5. Mix thoroughly on a vertical rotator at ~20 rpm for 10 minutes (or invert 20–30 times until fully emulsified).
6. Transfer the mixture to 2 mL or 15 mL Phase Lock Gel tubes (recommended for clean phase separation).
7. Centrifuge at 16,000 × g, 15 °C for 15 minutes.

• If not using phase-lock tubes:
• Incubate the tube on ice for 2 minutes until the organic layer becomes cloudy to stabilize the interphase.

• Avoid disturbing the interphase.
• Expected recovered volume: ~2.0 mL.

• Do not overdry; pellet becomes hard to dissolve

• For libraries prepared using DpnII, proceed directly to size selection.
• For libraries prepared using HindIII, the longer DNA fragments require an additional shearing step before size selection. Shear the 3C DNA to approximately 10 kb using a g-TUBE (Covaris, 520104), following the manufacturer's protocol.

• ~>4-5 kb size selection and sequencing on Revio / Sequel II.

• Make a 35% v/v dilution of AMPure PB beads by adding 1.75 mL of resuspended AMPure PB beads to 3.25 mL of elution buffer. The 35% dilution can be stored at 4°C for 30 days

1. Add 3.1X volume of diluted beads to the sample.
2. Mix thoroughly and spin briefly.
3. Let the sample sit for 10 minutes at room temperature to bind DNA.
4. Place on a magnetic rack and remove the supernatant.
5. Wash beads twice with 200 μL of 80% ethanol.
6. Dry beads as before and resuspend in 50 μL of elution buffer.
7. Incubate for 5 minutes, then separate beads on the magnet.
8. Transfer the supernatant to a new tube for sequencing or storage.

• After size selection, assess library quality by running undigested, digested, and 3C ligation products on a 1% agarose gel. Include a DNA ladder for size reference.
• Successful digestion and ligation typically produce a broad smear corresponding to fragmented and ligated chromatin.
• Optionally, analyze 3C DNA using automated pulsed-field capillary electrophoresis (e.g., Femto Pulse) to confirm the expected fragment size distribution.
• Representative gel images and fragment analyzer profiles are shown below.

Representative agarose gel showing restriction digestion efficiency. Undigested DNA, enzyme-digested DNA (DpnII or HindIII), and 3C ligation products are shown alongside a 1 kb ladder. Successful digestion produces a smear consistent with fragmented chromatin.

• Skip the gDNA shearing step for DpnII since we already digested it with the restriction enzyme, perform only for HindIII libraries. 
• Always use wide-bore tips to prevent damage to the DNA.
• Ensure reagents are thawed and mixed as directed. 

• 4 μL Repair Buffer
• 1 μL End Repair Mix
• 0.5 μL DNA Repair Mix
• 24.5 μL Digested 3C DNA Sample
• Total Volume: 30.0 μL

1. Mix the Repair Master Mix gently.
2. Briefly spin down the mix to collect liquid.
3. Add 5.5 µL Repair Master Mix to 24.5 µL DNA (final 30 µL).
4. Gently mix the samples and spin briefly to collect contents.
5. Set up the thermocycler with the following program:

• 30 minutes at 37°C
• 5 minutes at 65°C
• Hold at 4°C

• 2 μL Twist universal adapter
• 10 μL Ligation Mix
• 0.5 μL Ligation Enhancer
• 30 μL End-Repaired Sample
• Total Volume: 42.5 μL

1. Gently mix the Ligation Master Mix.
2. Spin briefly to collect the liquid.
3. Add 12.5 µL ligation master mix (Twist universal adapter + ligation reagents) to the 30 µL end-repaired sample (final 42.5 µL).
4. Mix samples gently and spin briefly.

• 30 minutes at 20°C
• Hold at 4°C

1. Add 42.5 μL of room-temperature beads to the samples.
2. Mix beads with the samples thoroughly.
3. Spin briefly to collect liquid.
4. Let the samples sit at room temperature for 10 minutes to bind DNA.
5. Place samples on a magnetic rack until beads separate.
6. Remove the supernatant carefully without disturbing the beads.
7. Add 200 μL of 80% ethanol to the beads, wait 30 seconds, and remove ethanol.
8. Repeat the ethanol wash step.
9. Dry the beads by:

• Removing residual ethanol while on the magnet.
• Briefly spinning and placing back on the magnet.

• 50 μL 2x Xtreme Buffer
• 20 μL dNTPs (2 mM each)
• 4 μL Twist UDI primers (plate)
• 2 μL KOD Xtreme Hot Start DNA Polymerase
• 24 μL Adapter-Ligated Sample
• Total Volume: 100 μL

1. Mix the PCR reagents thoroughly.
2. Spin briefly to collect the liquid.
3. Add 76 μL of PCR mix to each sample, ensuring a final volume of 100 μL.
4. Mix samples and spin briefly.

• 94 °C — 2 min (1 cycle)
• 98 °C — 10 sec (10-12 cycles)
• 58.8 °C — 30 sec (10-12 cycles)
• 68 °C — 10 min (10-12 cycles)
• 68 °C — 7 min (1 cycle)
• Hold at 4°C

1. Add 100 μL of beads to the samples.
2. Mix thoroughly and spin briefly.
3. Let the samples sit for 10 minutes at room temperature to bind DNA.
4. Place on a magnetic rack and remove supernatant.
5. Wash beads twice with 200 μL of 80% ethanol, waiting 30 seconds each time before removing ethanol.
6. Dry beads as before and resuspend in 50 μL of elution buffer.
7. Incubate for 5 minutes, then separate beads on the magnet.
8. Transfer the supernatant to a new tube for the next step.

• 500-600 ng input or more for Revio or Vega sequencing
• 250 ng input or more for Sequel II/IIe sequencing

• 8 μL Repair Buffer
• 2 μL End Repair Mix
• 1 μL DNA Repair Mix
• 49 μL Amplified DNA
• Total Volume: 60.0 μL

1. Mix the Repair Master Mix gently to combine.
2. Spin briefly to ensure all liquid is collected at the bottom.
3. Add 11 μL of the mix to each sample for a final volume of 60 μL.
4. Mix gently and spin briefly to collect liquid.
5. Use the thermocycler with the following program:

• 30 minutes at 37°C
• 5 minutes at 65°C
• Hold at 4°C

• 4 μL SMRTbell Adapter*
• 15 μL Ligation Mix
• 1 μL Ligation Enhancer
• 60 μL Repaired Amplified DNA
• Total Volume: 80.0 μL

1. Mix the ligation components thoroughly.
2. Spin briefly to collect liquid.
3. Add the prepared mix to the repaired DNA sample.
4. Mix gently and spin briefly.
5. Use the thermocycler with this program:

• 30 minutes at 20°C
• Hold at 4°C

1. Add 80 μL of cleanup beads to the sample.
2. Mix thoroughly and spin briefly.
3. Let the sample sit for 10 minutes at room temperature to bind DNA.
4. Place on a magnetic rack and remove the supernatant.
5. Wash beads twice with 200 μL of 80% ethanol, waiting 30 seconds each time before removing ethanol.
6. Dry beads as before and resuspend in 40 μL of elution buffer.
7. Incubate for 5 minutes, then separate beads on the magnet.
8. Transfer the supernatant to a new tube for the next step.

• 5 μL Nuclease Buffer
• 5 μL Nuclease Mix
• 40 μL SMRTbell Ligated DNA
• Total Volume: 50.0 μL

1. Mix the nuclease components thoroughly.
2. Spin briefly to collect liquid.
3. Add the nuclease mix to the ligated DNA sample.
4. Mix gently and spin briefly.
5. Use the thermocycler with this program:

• 15 minutes at 37°C
• Hold at 4°C

1. Add 50 μL of cleanup beads to the sample.
2. Mix thoroughly and spin briefly.
3. Let the sample sit for 10 minutes at room temperature to bind DNA.
4. Place on a magnetic rack and remove the supernatant.
5. Wash beads twice with 200 μL of 80% ethanol, waiting 30 seconds each time before removing ethanol.
6. Dry beads as before and resuspend in 50 μL of elution buffer.
7. Incubate for 5 minutes, then separate beads on the magnet.
8. Transfer the supernatant to a new tube for the next step.

•  >5 kb size selection and sequencing on Revio / Sequel II.

Option B: 35% diluted AMPure PB method (3.1×, removes <~3–5 kb)

• ~3-5 kb size selection and sequencing on Revio / Sequel II.

1. Prepare 35% AMPure PB beads by diluting with elution buffer.

• Make a 35% v/v dilution of AMPure PB beads by adding 1.75 mL of resuspended AMPure PB beads to 3.25 mL of elution buffer. The 35% dilution can be stored at 4°C for 30 days
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1. Add 3.1X volume of diluted beads to the sample.
2. Mix thoroughly and spin briefly.
3. Let the sample sit for 10 minutes at room temperature to bind DNA.
4. Place on a magnetic rack and remove the supernatant.
5. Wash beads twice with 200 μL of 80% ethanol.
6. Dry beads as before and resuspend in 50 μL of elution buffer.
7. Incubate for 5 minutes, then separate beads on the magnet.
8. Transfer the supernatant to a new tube for sequencing or storage.

Verification of fragment size during CiFi library preparation. Fragment size distributions were analyzed at key stages of the CiFi library preparation protocol using the Femto Pulse system (Agilent Technologies) for both DpnII (left column) and HindIII (right column) digests. The rows correspond to the following steps in the protocol: (A) 3C DNA QC, (B) After amplification, (C) After SMRTbell library preparation, and (D) After BluePippin size selection.