An improved method of constructing degradome library suitable for sequencing using Illumina platform
Abstract Background Post-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts ca...
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doaj-2a6af2d59a444785b03782a4ec72df5b2020-11-25T04:03:47ZengBMCPlant Methods1746-48112019-11-0115111210.1186/s13007-019-0524-7An improved method of constructing degradome library suitable for sequencing using Illumina platformYong-Fang Li0Miao Zhao1Menglei Wang2Junqiang Guo3Li Wang4Jie Ji5Zongbo Qiu6Yun Zheng7Ramanjulu Sunkar8College of Life Sciences, Henan Normal UniversityCollege of Life Sciences, Henan Normal UniversityCollege of Life Sciences, Henan Normal UniversityFaculty of Information Engineering and Automation, Kunming University of Science and TechnologyCollege of Life Sciences, Henan Normal UniversityCollege of Life Sciences, Henan Normal UniversityCollege of Life Sciences, Henan Normal UniversityFaculty of Information Engineering and Automation, Kunming University of Science and TechnologyDepartment of Biochemistry and Molecular Biology, Oklahoma State UniversityAbstract Background Post-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts can be identified by analyzing the degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries using Illumina platform, a widely used platform, is not so straightforward. Moreover, the currently used degradome or PARE methods utilize MmeI restriction site in the 5′ RNA adapter and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the members of the same target gene family or distinguishing miRNA biogenesis intermediates from the primary miRNA transcripts belonging to the same miRNA family. Consequently, developing a method which can generate longer fragments from the PARE or degradome libraries which can also be sequenced easily using Illumina platform is ideal. Results In this protocol, 3′ end of the 5′RNA adaptor of TruSeq small RNA library is modified by introducing EcoP15I recognition site. Correspondingly, the double-strand DNA (dsDNA) adaptor sequence is also modified to suit with the ends generated by the restriction enzyme EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation, thus amenable for sequencing using Illumina platform, like small RNA library. Conclusions Degradome library generated using this improved protocol can be sequenced easily using Illumina platform, and the resulting tag length is ~ 27-nt, which is longer than the MmeI generated fragment (20-nt) that can facilitate better accuracy in validating target transcripts belonging to the same gene family or distinguishing miRNA biogenesis intermediates of the same miRNA family. Furthermore, this improved method allows pooling and sequencing degradome libraries and small RNA libraries simultaneously using Illumina platform.http://link.springer.com/article/10.1186/s13007-019-0524-7CleavageDegradomeIllumina sequencingmiRNATarget gene |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yong-Fang Li Miao Zhao Menglei Wang Junqiang Guo Li Wang Jie Ji Zongbo Qiu Yun Zheng Ramanjulu Sunkar |
spellingShingle |
Yong-Fang Li Miao Zhao Menglei Wang Junqiang Guo Li Wang Jie Ji Zongbo Qiu Yun Zheng Ramanjulu Sunkar An improved method of constructing degradome library suitable for sequencing using Illumina platform Plant Methods Cleavage Degradome Illumina sequencing miRNA Target gene |
author_facet |
Yong-Fang Li Miao Zhao Menglei Wang Junqiang Guo Li Wang Jie Ji Zongbo Qiu Yun Zheng Ramanjulu Sunkar |
author_sort |
Yong-Fang Li |
title |
An improved method of constructing degradome library suitable for sequencing using Illumina platform |
title_short |
An improved method of constructing degradome library suitable for sequencing using Illumina platform |
title_full |
An improved method of constructing degradome library suitable for sequencing using Illumina platform |
title_fullStr |
An improved method of constructing degradome library suitable for sequencing using Illumina platform |
title_full_unstemmed |
An improved method of constructing degradome library suitable for sequencing using Illumina platform |
title_sort |
improved method of constructing degradome library suitable for sequencing using illumina platform |
publisher |
BMC |
series |
Plant Methods |
issn |
1746-4811 |
publishDate |
2019-11-01 |
description |
Abstract Background Post-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts can be identified by analyzing the degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries using Illumina platform, a widely used platform, is not so straightforward. Moreover, the currently used degradome or PARE methods utilize MmeI restriction site in the 5′ RNA adapter and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the members of the same target gene family or distinguishing miRNA biogenesis intermediates from the primary miRNA transcripts belonging to the same miRNA family. Consequently, developing a method which can generate longer fragments from the PARE or degradome libraries which can also be sequenced easily using Illumina platform is ideal. Results In this protocol, 3′ end of the 5′RNA adaptor of TruSeq small RNA library is modified by introducing EcoP15I recognition site. Correspondingly, the double-strand DNA (dsDNA) adaptor sequence is also modified to suit with the ends generated by the restriction enzyme EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation, thus amenable for sequencing using Illumina platform, like small RNA library. Conclusions Degradome library generated using this improved protocol can be sequenced easily using Illumina platform, and the resulting tag length is ~ 27-nt, which is longer than the MmeI generated fragment (20-nt) that can facilitate better accuracy in validating target transcripts belonging to the same gene family or distinguishing miRNA biogenesis intermediates of the same miRNA family. Furthermore, this improved method allows pooling and sequencing degradome libraries and small RNA libraries simultaneously using Illumina platform. |
topic |
Cleavage Degradome Illumina sequencing miRNA Target gene |
url |
http://link.springer.com/article/10.1186/s13007-019-0524-7 |
work_keys_str_mv |
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