Construction of High-Quality Rice Ribosome Footprint Library
High-throughput sequencing of ribosome footprints precisely maps and quantifies in vivo mRNA translation. The ribosome footprint sequencing has undergone continuing development since its original report. Here we provide a detailed protocol for construction of high-quality ribosome footprint library...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-09-01
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| Series: | Frontiers in Plant Science |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/article/10.3389/fpls.2020.572237/full |
| _version_ | 1819115986525093888 |
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| author | Xiaoyu Yang Jie Cui Bo Song Yu Yu Beixin Mo Lin Liu |
| author_facet | Xiaoyu Yang Jie Cui Bo Song Yu Yu Beixin Mo Lin Liu |
| author_sort | Xiaoyu Yang |
| collection | DOAJ |
| description | High-throughput sequencing of ribosome footprints precisely maps and quantifies in vivo mRNA translation. The ribosome footprint sequencing has undergone continuing development since its original report. Here we provide a detailed protocol for construction of high-quality ribosome footprint library of rice. Rice total polysomes are isolated with a modified low ionic polysome extraction buffer. After nuclease digestion, rice ribosome footprints are extracted using SDS method followed by column purification. High-quality rice ribosome footprint library with peak reads of approximately 28-nucleotide (nt) length and strong 3-nt periodicity is constructed via key steps including rRNA depletion, end repair, 3’ adapter ligation, reverse transcription, circularization, PCR enrichment and several rounds of purification. Biological significance of rice ribosome footprint library is further revealed by the comparison of transcriptomic and translatomic responses to salt stress and the utilization for novel open reading frame (ORF) identification. This improved protocol for rice ribosome footprint library construction will facilitate the global comprehension and quantitative measurement of dynamic translation in rice. |
| first_indexed | 2024-12-22T05:09:54Z |
| format | Article |
| id | doaj.art-cad6f11d04624375b2dfcfcd17ca6721 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-22T05:09:54Z |
| publishDate | 2020-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-cad6f11d04624375b2dfcfcd17ca67212022-12-21T18:38:00ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2020-09-011110.3389/fpls.2020.572237572237Construction of High-Quality Rice Ribosome Footprint LibraryXiaoyu Yang0Jie Cui1Bo Song2Yu Yu3Beixin Mo4Lin Liu5Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Longhua Bioindustry and Innovation Research Institute, Shenzhen University, Shenzhen, ChinaGuangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Longhua Bioindustry and Innovation Research Institute, Shenzhen University, Shenzhen, ChinaShenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, ChinaGuangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Longhua Bioindustry and Innovation Research Institute, Shenzhen University, Shenzhen, ChinaGuangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Longhua Bioindustry and Innovation Research Institute, Shenzhen University, Shenzhen, ChinaGuangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Longhua Bioindustry and Innovation Research Institute, Shenzhen University, Shenzhen, ChinaHigh-throughput sequencing of ribosome footprints precisely maps and quantifies in vivo mRNA translation. The ribosome footprint sequencing has undergone continuing development since its original report. Here we provide a detailed protocol for construction of high-quality ribosome footprint library of rice. Rice total polysomes are isolated with a modified low ionic polysome extraction buffer. After nuclease digestion, rice ribosome footprints are extracted using SDS method followed by column purification. High-quality rice ribosome footprint library with peak reads of approximately 28-nucleotide (nt) length and strong 3-nt periodicity is constructed via key steps including rRNA depletion, end repair, 3’ adapter ligation, reverse transcription, circularization, PCR enrichment and several rounds of purification. Biological significance of rice ribosome footprint library is further revealed by the comparison of transcriptomic and translatomic responses to salt stress and the utilization for novel open reading frame (ORF) identification. This improved protocol for rice ribosome footprint library construction will facilitate the global comprehension and quantitative measurement of dynamic translation in rice.https://www.frontiersin.org/article/10.3389/fpls.2020.572237/fullOryza sativahigh-qualityhigh-throughput sequencingribosome footprint librarytranslation |
| spellingShingle | Xiaoyu Yang Jie Cui Bo Song Yu Yu Beixin Mo Lin Liu Construction of High-Quality Rice Ribosome Footprint Library Frontiers in Plant Science Oryza sativa high-quality high-throughput sequencing ribosome footprint library translation |
| title | Construction of High-Quality Rice Ribosome Footprint Library |
| title_full | Construction of High-Quality Rice Ribosome Footprint Library |
| title_fullStr | Construction of High-Quality Rice Ribosome Footprint Library |
| title_full_unstemmed | Construction of High-Quality Rice Ribosome Footprint Library |
| title_short | Construction of High-Quality Rice Ribosome Footprint Library |
| title_sort | construction of high quality rice ribosome footprint library |
| topic | Oryza sativa high-quality high-throughput sequencing ribosome footprint library translation |
| url | https://www.frontiersin.org/article/10.3389/fpls.2020.572237/full |
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