Jun 10, 2025

Public workspaceCycloneSEQ long-read RNA-seq Library Preparation and Sequencing Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwq3o7vmk/v1
CycloneSEQ long-read RNA-seq Library Preparation and Sequencing Protocol
  • Qunfei Guo1,
  • youliang Pan1,
  • Wei Dai1,
  • Wei Jiang1
  • 1BGI
  • GigaScience Press
  • BGI
shi shuaizhen
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwq3o7vmk/v1
Protocol Citation: Qunfei Guo, youliang Pan, Wei Dai, Wei Jiang 2025. CycloneSEQ long-read RNA-seq Library Preparation and Sequencing Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwq3o7vmk/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: 219762
Keywords: CycloneSeq, Long-read Sequencing, RNA-seq, Genomics, Transcriptomics, animal rna on the cycloneseq platform, cycloneseq platform, cycloneseq, length transcriptome library preparation, read rna, extracted animal rna, transcriptome, rna, seq library preparation, sequencing protocol, sequencing, sequencing protocol this protocol, seq
Abstract
This protocol is designed for full-length transcriptome library preparation and sequencing of extracted animal RNA on the CycloneSEQ platform.
Troubleshooting
Genomic DNA Removal
Treat 100–500 ng total RNA with 5× gDNA Digester Mix in a nuclease-free tube.
  • Incubate at 42°C for 2 min.
  • Centrifuge briefly to collect liquid.
First-Strand cDNA Synthesis
Prepare 20 μL reaction mix containing:
  • 4× Hifair SuperMix
  • Random Primers (50 μM)
  • Treated RNA template

Incubate at 72°C for 5 min.
  • Heat-inactivate at 85°C for 5 sec.

Note: For high-GC RNA templates, increase incubation temperature to 60°C.
CycloneSEQ Library Preparation
Process cDNA into sequencing libraries using the CycloneSEQ Universal Library Prep Set:
  • DNA Repair (end-repair/dA-tailing)
  • Adapter Ligation (CycloneSEQ-specific adapters)

Follow the standard whole-genome sequencing workflow steps provided in the kit.
Library Quality Control
Quantify libraries using a Qubit fluorometer.

Assess fragment size distribution (e.g., using Agilent TapeStation).
Sequencing
Load libraries onto the CycloneSEQ WuTong02 platform.

Run sequencing using manufacturer-recommended parameters (e.g., 48-hr run, >Q20 accuracy).