Mar 19, 2026

Public workspaceArima 3C with PacBio HiFi Sequencing (CiFi) Library Preparation Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l2zyrpl1y/v1
  • Gulhan Kaya1,
  • Sean P. McGinty1,
  • Mohamed Abuelanin1,
  • Megan Y. Dennis1
  • 1Genome Center, MIND Institute, and Department of Biochemistry & Molecular Medicine, University of California, Davis, CA 95616, USA
  • Dennis Lab_UC Davis Genome Center
Megan Y. Dennis
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.4r3l2zyrpl1y/v1
Protocol Citation: Gulhan Kaya, Sean P. McGinty, Mohamed Abuelanin, Megan Y. Dennis 2026. Arima 3C with PacBio HiFi Sequencing (CiFi) Library Preparation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2zyrpl1y/v1
Manuscript citation:
McGinty, S.P., Kaya, G., Sim, S.B. et al. CiFi: accurate long-read chromosome conformation capture with low-input requirements. Nat Commun 17, 215 (2026). https://doi.org/10.1038/s41467-025-66918-y
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 10, 2026
Last Modified: March 19, 2026
Protocol Integer ID: 242943
Keywords: arima 3c with pacbio hifi sequencing, pacbio hifi sequencing, chromatin conformation capture with pacbio hifi, arima genomic, fragment length distribution for pacbio platform, read sequencing, pacbio platform, chromatin interaction library, read chromatin interaction library, arima 3c, version of the cifi workflow, pacbio hifi, cifi workflow, library preparation protocol this protocol, chromatin conformation capture, library preparation protocol, cifi, conversion to smrtbell library, dna purification
Abstract
This protocol describes Arima-3C–CiFi, a workflow that integrates Arima Genomics 3C chromatin conformation capture with PacBio HiFi sequencing to generate long-read chromatin interaction libraries. The method includes cell crosslinking, restriction digestion, proximity ligation, DNA purification, and conversion to SMRTbell libraries for long-read sequencing. Optional size selection and quality control steps are included to optimize fragment length distribution for PacBio platforms.
This protocol represents an amended and updated version of the CiFi workflow compared with the originally published protocol.
Materials
User Supplied Equipment, Reagents and Consumables:

- 37% Formaldehyde (e.g. Fisher Scientific® Cat # F79-500)
- 100% Ethanol and freshly prepared 80% and 70% Ethanol
- Phenol:Chloroform:Isoamyl Alcohol (25:24:1 v/v; Thermo Fisher Cat # 15593031)
- 3M sodium acetate pH 5.2 (VWRVE521-100ML)
- Glycogen (optional) (20 µg/µL; Thermo Fisher Cat # 10814010)
- 1X PBS, pH 7.4 (e.g. Fisher Scientific® Cat # 50-842-949)
- Qubit® Fluorometer, dsDNA HS Assay Kit and consumables (e.g. Thermo Fisher Scientific Cat # 32851, 32856)
- 15mL conical tubes
- 1.7 mL DNA LoBind tubes, PCR tubes, or PCR plates, and a magnetic rack compatible with the selected tubes.
- Centrifuge
- Thermal cycler (if performing Arima-3C Protocol in PCR tubes or PCR plate)
- Thermomixer (if performing Arima-3C Protocol in 1.7mL microcentrifuge tubes)
- Phase Lock Gel tubes
- Wide-bore tips
- Low TE buffer (pH 8.0, Cat# 102-178-400)

Required kits:

- Arima-3C Kit (cat# A101050; User Guide; Doc A160182 v02)
- AMPure® XP Beads (Beckman Coulter Cat # A63880) - SMRTbell prep kit 3.0, cat# 102-182-700
https://www.pacb.com/wp-content/uploads/Insert-SMRTbell-prep-kit-3.0.pdf
- Optional: SMRTbell adapter index plate 96A, cat# 102-009-200 (SMRTbell Adapter Index Plate 96A for multiplexing instead of the Overhang Adapter V3)
https://www.pacb.com/wp-content/uploads/Insert-SMRTbell-adapter-index-plate-96A.pdf
- SMRTbell cleanup beads, cat# 102-158-300
https://www.pacb.com/wp-content/uploads/Insert-SMRTbell-Cleanup-Beads.pdf - KOD Xtreme Hot Start DNA Polymerase, Sigma Aldrich cat# 71975-M
https://www.sigmaaldrich.com/US/en/product/mm/71975m
- AMPure PB bead size selection kit, cat# PacBio 10-2-182-500
- Twist Universal Adapter System, cat# Twist 101307-101311
https://www.pacb.com/wp-content/uploads/Procedure-checklist-Amplifying-genomic-DNA-for-SMRTbell-library-preparation-and-HiFi-sequencing.pdf
Troubleshooting
Safety warnings
Enzyme H1 storage
  • Store at –80°C
  • Storage at –20°C reduces activity after ~3 months

Conditioning Solution & Buffer D
  • May form precipitates
  • Dissolve by warming at 37°C for 5–15 min if needed

Reagent handling
  • Keep Box A reagents at room temperature
  • Keep Box B reagents on ice

  • Exception: Enzyme D store on ice but warm to RT just before use

Viscous reagents
  • Some Box A reagents are viscous
  • Pipette slowly and carefully
Before start
  • The Arima-3C protocol for mammalian cell lines begins with harvesting and crosslinking 5–10 million cells.
  • Arima-3C reactions perform optimally with crosslinked material corresponding to ~3–4 µg of DNA per reaction.
  • If the number of crosslinked cells or DNA yield is unknown, complete the Estimating Input Amount protocol to determine DNA yield per 1×10⁶ cells and calculate the appropriate input.
  • The Arima-3C kit provides sufficient reagents for up to 8 samples.

Input Requirements
  • The crosslinked cell pellet for one reaction should occupy ≤20 µL and be free of residual liquid.
  • If the pellet volume exceeds 20 µL, split the sample so that each reaction contains ≤20 µL pellet volume and total DNA input per reaction remains ~3–4 µg
Part 1: Arima-3C Library Preparation
This section describes preparation of the Arima 3C library from crosslinked cells.
  • 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.
Step 1 — Crosslinking (Standard or low Input)
Input: Cultured mammalian cells Output: Crosslinked cell pellets

Standard Input
  1. Harvest cells using standard cell culture procedures and pellet by centrifugation.
  2. Resuspend cells in culture media and determine cell count using a hemocytometer or automated counter.
  3. Transfer 5–10 million cells to a new 15-mL conical tube, pellet, and remove supernatant.
  4. Resuspend cells in 5 mL RT 1× PBS.
  5. Add 286 µL of 37% formaldehyde to reach 2% final concentration. Note: If using 16% formaldehyde, add 714 µL instead.
  6. Mix by gentle inversion (10×) and incubate 10 min at RT.
  7. Add 460 µL Stop Solution 1, invert 10×, incubate 5 min at RT. Note: If using 16% formaldehyde, add 497 µL Stop Solution 1.
  8. Incubate on ice 15 min.
  9. Pellet cells and discard supernatant.
  10. Wash by resuspending in 5 mL 1× PBS.
  11. Aliquot into tubes containing 1 × 10⁶ cells each.
  12. Pellet aliquots and remove all residual liquid.
  13. Snap-freeze on dry ice or liquid nitrogen.
  14. Store at –80°C until use.

Low Input (For fewer than 1 million cells)
  1. Collect cells in a 1.7 mL microfuge tube, pellet cells (e.g., 500×g, 5 min, RT), and remove supernatant.
  2. Add 1 mL 1× PBS (RT) + 3% BSA (w/v) and mix by inversion 5×.
  3. Add 57 µL 37% formaldehyde (final ~2%), invert 10×, and incubate 10 min at RT, inverting occasionally.
  4. Add 91.9 µL Stop Solution 1, invert 10×, and incubate 5 min at RT, inverting occasionally.
  5. Place on ice 15 min.
  6. Pellet cells (e.g., 500×g, 5 min, 4 °C or RT) and remove supernatant.
  7. Add 1 mL 1× PBS (RT) + 3% BSA (w/v) and invert 5×.
  8. Pellet cells again and remove supernatant.
  9. Remove residual liquid completely, leaving a clean crosslinked pellet.
  10. Snap-freeze on dry ice or liquid nitrogen.
  11. Store at –80°C until use.
Step 2 — Estimating Input Amount (Optional but Recommended)
Purpose: Determine DNA yield per 1 × 10⁶ cells to calculate optimal input.
  1. Thaw one aliquot (1 × 10⁶ cells).
  2. Add 209.5 µL master mix:
  • 174 µL Elution Buffer
  • 10.5 µL Buffer D
  • 25 µL Enzyme D

3. Add 20 µL Buffer E and incubate:
  • 55°C — 30 min
  • 68°C — 90 min
  • 4°C — hold

4. Purify using AMPure XP beads (150 µL).
5. Elute in 20 µL Elution Buffer.
6. Quantify using Qubit dsDNA HS assay.
7. Calculate the number of cells needed to obtain ~3–4 µg DNA per Arima reaction.
Step 3 — Arima-3C Reaction
Purpose: Generate proximally ligated 3C DNA from crosslinked cells.

Input: Crosslinked cells containing ~3–4 µg DNA Output: Arima-3C DNA (Proximally ligated DNA)
Cell Lysis

- Resuspend one reaction of crosslinked cells in 20 µL Lysis Buffer in a tube or PCR plate well. - Incubate at 4 °C for 15 min.
Chromatin Conditioning

- Add 24 µL Conditioning Solution. - Mix gently by pipetting. - Incubate at 62 °C for 10 min.
If using a thermal cycler, set the lid temperature to 85 °C.
Stop Reaction

- Add 20 µL Stop Solution 2.
- Mix gently by pipetting. - Incubate at 37 °C for 15 min.
If using a thermal cycler, set lid temperature to 85 °C.
Restriction Enzyme Digestion

Prepare the following master mix per reaction:
  • 11 µL Water
  • 9 µL Buffer H
  • 8 µL Enzyme H Total: 28 µL
Add 28 µL to each reaction.

Note: Prepare reagents as a master mix before addition.
Sequential Incubations

Mix gently by pipetting and incubate sequentially:
  • 37 °C — 60 min
  • 65 °C — 20 min
  • 25 °C — 10 min
If using a thermal cycler, set lid temperature to 85 °C.

Optional: The 37 °C incubation can be extended or held overnight if needed.
Post-Digestion Mix and QC Aliquot

Mix gently by inversion.
- It is normal to see white particles in solution, which are nuclei/cells.
- Immediately transfer 10 µL to a new tube labeled “Digestion QC.”
(The Digestion QC aliquot is used to verify digestion efficiency and chromatin fragmentation prior to downstream processing.)
- Store at –20 °C for later QC analysis.

Proceed with the remaining sample.

Add Enzyme C Master Mix
Per reaction:
  • 10 µL Water
  • 70 µL Buffer C
  • 12 µL Enzyme C Total: 92 µL
Add 92 µL and mix gently.
Incubate at room temperature for 15 min.

Note: Enzyme D should be equilibrated to room temperature before use.
Add Enzyme D Master Mix
Per reaction:
  • 10.5 µL Buffer D
  • 25 µL Enzyme D Total: 35.5 µL
Add and mix gently.
Add Buffer E and Incubate
Add 20 µL Buffer E, mix gently by pipetting, and incubate:
  • 55 °C — 30 min
  • 68 °C — 90 min
  • 25 °C — 10 min
If using a thermal cycler, set lid temperature to 85 °C.

Important: Do not exceed 90 min at 68 °C unless using a heated lid.
To provide flexibility, the 25 °C step may be replaced with an overnight hold at 4 °C.
Proceed to DNA Purification
Choose one of the following:

Phenol–Chloroform Extraction (recommended) More labor-intensive and involves hazardous chemicals, but may improve DNA purity. This method has been used in published Pore-C workflows.
or
SPRI Bead Purification Simpler magnetic bead-based cleanup. May result in slightly lower DNA purity, but typically yields comparable DNA amounts.

Both methods generally produce similar total DNA yields.
Step 4 —DNA Purification
Phenol–Chloroform Extraction
  1. Cool the sample on ice after completing the reverse cross-linking incubation.
  2. Transfer the entire sample to a DNA LoBind centrifuge tube.
  3. Add an equal volume of chilled phenol:chloroform:isoamyl alcohol (25:24:1). Perform in a fume hood.
  4. Mix thoroughly on a vertical rotator at ~20 rpm for 10 minutes (or invert 20–30 times until fully emulsified).
  5. Transfer the mixture to 2 mL or 15 mL Phase Lock Gel tubes (recommended for clean phase separation).
  6. 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.
8. Carefully transfer the aqueous (upper) phase to a new 5 mL DNA LoBind tube or a 15 mL centrifuge tube.
  • Avoid disturbing the interphase.
  • Expected recovered volume: ~2.0 mL.
Ethanol Precipitation
9. Add 0.1 volumes of 3 M sodium acetate pH 5.2
10. Mix by gentle inversion. The solution may briefly turn cloudy.
11. Add ~3 volumes of 100% ethanol
12. Mix gently by inversion 10–15 times.
13. Precipitate DNA at –80 °C for at least 1 hour or –20 °C overnight (recommended for best recovery).
14. Pre-cool centrifuge to 4 °C.
15. Centrifuge samples at 16,000 × g, 4 °C for 30 minutes.
16. Carefully discard supernatant without disturbing pellet.
17. Wash pellet with 4 mL of 80% ethanol (or 2 mL if using 2 mL tubes).
18. Centrifuge at 16,000 × g, 4 °C for 5 minutes.
19. Discard supernatant, then wash with 2 mL of 70% ethanol.
20. Centrifuge again at 16,000 × g, 4 °C for 5 minutes.
21. Remove supernatant completely. Briefly spin and remove remaining droplets.
22. Air-dry pellet for ~3-5 minutes only.
  • Do not overdry; pellet becomes hard to dissolve
23. Resuspend each pellet in 52 µL of TE buffer (pH 8.0).
24. If multiple tubes were used, dissolve the first pellet and transfer the solution onto the next pellet until all are combined.
25. Incubate at room temperature for 5 minutes, gently inverting every few minutes.
26. Briefly spin down.
27. DNA can be pooled into a single 1.5 mL DNA LoBind tube.
28. Store at 4°C short-term or –20°C long-term.
29. Quantify DNA using Qubit DNA HS assay.
30. Proceed to the next CiFi workflow steps (size selection, 3C DNA QC, etc.)
Step 5 —Size Selection
Purpose: Enrich for long DNA fragments suitable for PacBio HiFi sequencing.
Choose one of the following size-selection methods depending on desired fragment cutoff.

Option 1 — Standard PacBio AMPure PB Cleanup (0.45× Ratio)
(Removes fragments <~3 kb)
  1. Add 0.45× volume AMPure PB beads to the 3C DNA sample.
  2. Mix thoroughly and incubate 10 min at room temperature.
  3. Place on a magnetic rack until clear (~5 min).
  4. Carefully remove and discard supernatant.
  5. Wash beads twice with 200 µL 80% ethanol while on the magnet.
  6. Do not overdry the beads, as this can reduce DNA recovery.
  7. Elute DNA in 47 µL Low TE (pH 8.0).
  8. Incubate 5 min, place on magnet, and transfer eluate to a new tube.

Option 2 — Diluted AMPure PB Bead Size Selection
(Removes fragments <~4–5 kb; recommended for Revio/Sequel II)
This method enriches for longer fragments.
Prepare 35% Beads
Prepare a 35% (v/v) bead dilution:
  • 1.75 mL AMPure PB beads
  • 3.25 mL Elution Buffer
Store at 4 °C for up to 30 days.

Size Selection

  1. Add 3.1× volume of diluted beads to the sample.
  2. Mix thoroughly and spin briefly.
  3. Incubate 10 min at room temperature.
  4. Place on magnet and remove supernatant.
  5. Wash beads twice with 200 µL 80% ethanol.
  6. Air-dry briefly (do not overdry).
  7. Resuspend in 50 µL elution buffer.
  8. Incubate 5 min, place on magnet.
  9. Transfer supernatant to a new tube.
Step 6 — QC
  • Run undigested, digested, and 3C products on a 1% agarose gel.
  • Analyze 0.5 ng/µL DNA using Femto Pulse.
  • Confirm expected fragment size distribution.


Quality check of Arima 3C DNA


Verification of Fragment Size for Arima 3C DNA QC

Part 2: Preparing HiFi SMRTbell CiFi Libraries
  • Always use wide-bore tips to prevent damage to the DNA.
  • Ensure reagents are thawed and mixed as directed. 

Note: KOD Xtreme can, in theory, be replaced with https://www.takarabio.com/products/pcr/long-range-pcr/primestar-longseq-dna-polymerase?catalog=R055A
I. Amplification
1. Repair and A-Tailing of digested 3C DNA
This step repairs DNA damage and adds A-tails in a single reaction to prepare for adapter ligation.
Repair Master Mix:
  • 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

Instructions:
  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

6. Move to the next step of the protocol.
2. Ligation of Linear Amplification Adapter and Cleanup
This step adds the amplification adapter to DNA fragments. Ensure cleanup beads are at room temperature before starting.

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

Instructions:
  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.
5. Use the thermocycler with the following program:
  • 30 minutes at 20°C
  • Hold at 4°C

Cleanup with 1X SMRTbell Cleanup Beads
  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.
10. Resuspend the beads in 24 μL of elution buffer.
11. Spin briefly, then incubate for 5 minutes at room temperature.
12. Place samples back on the magnet and transfer the supernatant to a new tube.
3. Amplification and Cleanup
Amplify DNA fragments with adapters on both ends and clean them using 1X SMRTbell Cleanup Beads.

PCR Master Mix:
  • 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

Instructions:
  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.
5. Use the thermocycler with this program:

  • 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

DNA Input and PCR Cycles: - 1 ng: 14 cycles
- 5 ng: 12 cycles - 10 ng: 11 cycles - 20 ng: 10 cycles
- 50 ng: 8 cycles

Cleanup with 1X SMRTbell Cleanup Beads
  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.
II. SMRTbell library prep
4. Repair and A-Tailing of Amplified DNA
This step prepares amplified DNA by repairing damage and adding A-tails for SMRTbell adapter ligation.
  • 500-600 ng input or more for Revio or Vega sequencing
  • 250 ng input or more for Sequel II/IIe sequencing

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

Instructions:
  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
6. Proceed to the next step in the workflow.
5. SMRTbell Adapter Ligation and Cleanup
This step ligates the SMRTbell adapter to the ends of the amplified DNA fragments.

Ligation Master Mix:
  • 4 μL SMRTbell Adapter*
  • 15 μL Ligation Mix
  • 1 μL Ligation Enhancer
  • 60 μL Repaired Amplified DNA
  • Total Volume: 80.0 μL
*Exclude the SMRTbell adapter if using the SMRTbell adapter index plate 96A

Instructions:
  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

Cleanup with 1X SMRTbell Cleanup Beads
  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 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.

6. Nuclease Treatment
This step removes un-ligated, semi-ligated, or damaged DNA fragments from the sample.

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

Instructions:
  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

Cleanup with SMRTbell Cleanup Beads (1X)
  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.
III. Size-Selection of SMRTbell Library and Sequencing
Size selection thresholds may be adjusted depending on sequencing platform and target fragment distribution.

Option A: BluePippin high-pass (≥5 kb)

  •  >5 kb size selection and sequencing on Revio / Sequel II.
  • Follow manufacturer instructions for cassette selection and size cutoff.

or

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


This step enriches the sample for DNA fragments of the desired size.
Instructions:
  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

  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.