Mar 15, 2024
PacBio Isoseq samples preparation
- 1Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, BMC A11, Lund University, 221 84 Lund, Sweden.
Protocol Citation: anita.adami 2024. PacBio Isoseq samples preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm25j6g3p/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: February 06, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 76484
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Swedish Research Council
Grant ID: 2018-02694
Swedish Brain Foundation
Grant ID: FO2019-0098
Cancerfonden
Grant ID: 190326
Barncancerfonden
Grant ID: PR2017-0053
NIHR Cambridge Biomedical Research Centre
Grant ID: NIHR203312
Swedish Society for Medical Research
Grant ID: S19-0100
National Institutes of Health
Grant ID: HG002385
Swedish Research Council
Grant ID: 2021-03494
Swedish Research Council
Grant ID: 2020-01660
Abstract
This protocol described how to prepare samples for PacBio Isoseq
RNA extraction
RNA extraction
The total RNA was obtained from brain tissue samples using miRNA Easy Mini Kit (Qiagen). The manufacturer's instructions were followed step-by-step.
RNA samples were subsequently put on dry ice and shipped to National Genomics Infrastructure of Sweden.
Samples QC evaluation
Samples QC evaluation
At the Nation Genomics Infrastructure of Sweden, input QC of samples was performed on the Agilent Bioanalyzer instrument, using the Eukaryote Total RNA Nano kit to evaluate RIN and concentration.
Libraries preparation
Libraries preparation
The sample libraries were prepared as described in "Procedure & Checklist – Iso-Seq™ Express Template Preparation for Sequel® and Sequel II Systems" (PN 101-763-800 Version 02 (October 2019)) using the NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module, the Iso-Seq Express Oligo Kit, ProNex beads (Promega) and the SMRTbell Express Template Prep Kit 2.0.
300 ng of total RNA was used for cDNA Synthesis followed by 12 + 3 cycles of cDNA Amplification.
In the purification step of amplified cDNA, the standard workflow was applied (sample is composed primarily of transcripts centered around 2 kb).
After purification, the amplified cDNA went into SMRTbell library construction.
Quality control of the SMRTbell libraries was performed with the Qubit dsDNA HS kit and the Agilent Bioanalyzer High Sensitivity kit.
Primer annealing and polymerase binding was performed using the Sequel II binding kit 2.0.
PacBio Isoseq sequencing
PacBio Isoseq sequencing
Finally, the samples were sequenced on Sequel II and Sequel IIe System using Sequel® II Sequencing Plate 2.0, On-Plate Loading Concentration of 110 pM, movie time 24 hours and pre-extension time 2 hours.