Jun 10, 2025
CycloneSEQ based Pore-C Library Preparation and Sequencing Protocol
- Qunfei Guo1,
- youliang Pan1,
- Wei Dai1,
- Wei Jiang1
- 1BGI
Protocol Citation: Qunfei Guo, youliang Pan, Wei Dai, Wei Jiang 2025. CycloneSEQ based Pore-C Library Preparation and Sequencing Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmmr5bv3p/v1
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: June 07, 2025
Last Modified: June 10, 2025
Protocol Integer ID: 219760
Keywords: CycloneSeq, Long-read sequencing, Genomics, animal dna on the cycloneseq, cycloneseq, sequencing platform, sequencing protocol, extracted animal dna, sequencing protocol this protocol, sequencing, dna, pore
Abstract
This protocol is designed for Pore-C library preparation and sequencing of extracted animal DNA on the CycloneSEQ sequencing platform.
Troubleshooting
Tissue Fixation & Crosslinking
Homogenization & Washing:
- Using 100 mg of fresh tissue per sample, homogenize in 2 mL 1x PBS using a sterile pestle.
- Centrifuge at 2,000xg for 5min at 4°C. Discard supernatant.
Fixation:
- Resuspend pellet in 10 mL 1% formaldehyde in 1x PBS
- Incubate 10 min at RT with gentle rotation.
Quenching
- Add 527 μL 2.5 M glycine.
- Incubate 5 min at RT, then incubate 10 min on ice.
- Centrifuge at 2,000xg for 10 min at 4°C. Discard the supernatant.
Nuclei isolation & Digestion
Lysis:
- Resuspend pellet in 550 μL ice-cold lysis buffer
(10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% lgepal-CA630, 50 μL of protease inhibitor cocktail)
- Incubate 15 min on ice.
- Centrifuge at 2,000xg for 10 min at 4°C. Discard supernatant.
Digestion Prep:
- Resuspend pellet in 200 μL ice-cold 1.5× digestion buffer.
- Centrifuge at 2,000×g for 10 min at 4°C.
- Resuspend pellet in 300 μL 1.5× digestion buffer.
- Add 33.5 μL 1% SDS.
Digestion:
- Incubate in thermomixer: 65°C, 300 rpm, 10 min.
- Add 37.5 μL 10% Triton X-100 (to quench SDS).
- Add 45 μL NlaIII (10 U/μL) + 34 μL nuclease-free water.
- Rotate 18 hr at 37°C.
- Heat-inactivate: 65°C for 20 min.
Proximity Ligation
Ligation Master Mix:
Prepare 550 μL mix per sample:
- 100 μL 10× T4 DNA ligase buffer (with ATP)
- 10 μL BSA (10 mg/mL)
- 50 μL T4 DNA ligase (400 U/μL)
- 390 μL nuclease-free water
Ligation:
- Add master mix to sample.
- Rotate 5 hr at RT.
Crosslink Reversal & DNA Purification
Protein Digestion:
- Add master mix:
(1) 100 μL 10% SDS
(2) 500 μL 20% Tween-20
(3) 100 μL Proteinase K (20 mg/mL)
(4) 300 μL nuclease-free water
- Incubate 56°C for 18 hr.
DNA Extraction:
- Add equal volume phenol:chloroform:isoamyl alcohol (25:24:1).
- Centrifuge 12,000×g for 15 min at 4°C.
- Transfer aqueous phase to new tube.
Precipitation:
- Add 0.7× volume isopropanol.
- Incubate -20°C for 1 hr.
- Centrifuge 12,000×g for 30 min at 4°C.
- Wash pellet with 70% ethanol. Air-dry.
Library Construction
Size Selection & Bead Cleanup:
- Resuspend DNA in 50 μL nuclease-free water.
- Perform size selection >1.5 kb using DNA Clean Beads per manufacturer’s instructions.
CycloneSEQ Library Prep:
- Construct library using CycloneSEQ Long-Read WGS Kit (refer to separate protocol).
- Quantify with Qubit fluorometer.
Sequencing
Run Sequencing:
- Load library onto CycloneSEQ WuTong02 platform.
- Follow manufacturer’s run parameters.