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Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples
Published on: June 8, 2020
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An improved method of constructing degradome library suitable for sequencing using Illumina platform.
Yong-Fang Li1, Miao Zhao1, Menglei Wang1
11College of Life Sciences, Henan Normal University, Xinxiang, Henan People's Republic of China.
Plant Methods
|December 14, 2019
Summary
This study introduces an improved degradome library preparation method for accurate microRNA target validation. The new protocol generates longer sequencing fragments, enhancing gene family and miRNA intermediate identification on the Illumina platform.
Area of Science:
- Molecular Biology
- Genomics
- Gene Regulation
Background:
- MicroRNAs (miRNAs) are key regulators of gene expression, mediating messenger RNA (mRNA) cleavage.
- Degradome or PARE sequencing identifies transcriptome-wide mRNA cleavage sites.
- Current methods using Illumina sequencing face challenges with short fragment lengths (20-nt) and distinguishing similar gene family members or miRNA intermediates.
Purpose of the Study:
- To develop an improved method for generating longer, easily sequenceable degradome libraries.
- To enhance the accuracy of identifying miRNA targets and distinguishing miRNA biogenesis intermediates.
Main Methods:
- Modification of the 5' RNA adaptor in TruSeq small RNA libraries with an EcoP15I recognition site.
- Adaptation of double-strand DNA (dsDNA) adaptor sequences for EcoP15I restriction enzyme compatibility.
- Utilizing primer pairs from small RNA library preparation for degradome library amplification.
Main Results:
- The improved protocol yields degradome library fragments of approximately 27-nt, longer than the 20-nt fragments from MmeI-based methods.
- This increased fragment length improves accuracy in validating target transcripts within the same gene family.
- The method facilitates better discrimination between miRNA biogenesis intermediates and primary miRNA transcripts of the same miRNA family.
Conclusions:
- The enhanced degradome library protocol is readily sequenced on the Illumina platform.
- Longer sequencing tags improve the precision of target transcript validation and miRNA intermediate identification.
- Simultaneous pooling and sequencing of degradome and small RNA libraries are enabled by this improved method.
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