Construction of High-Quality Rice Ribosome Footprint Library

文献类型： 外文期刊

第一作者： Yang, Xiaoyu

作者： Yang, Xiaoyu;Cui, Jie;Yu, Yu;Mo, Beixin;Liu, Lin;Song, Bo

作者机构：

关键词： Oryza sativa; high-quality; high-throughput sequencing; ribosome footprint library; translation

期刊名称：FRONTIERS IN PLANT SCIENCE （ 影响因子：5.753； 五年影响因子：6.612 ）

ISSN： 1664-462X

年卷期： 2020 年 11 卷

页码：

收录情况： SCI

摘要： High-throughput sequencing of ribosome footprints precisely maps and quantifiesin vivomRNA 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 constructedviakey 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.

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