核糖体分析
核糖体
核糖核酸酶P
生物
内部核糖体进入位点
计算生物学
5.8S核糖体RNA
核糖体RNA
闪耀达尔加诺序列
核酸酶
核糖核酸
细胞生物学
遗传学
基因
作者
Lucas Ferguson,Heather E. Upton,Sydney C. Pimentel,Amanda Mok,Liana F. Lareau,Kathleen L. Collins,Nicholas T. Ingolia
出处
期刊:Nature Methods
[Nature Portfolio]
日期:2023-10-02
卷期号:20 (11): 1704-1715
被引量:9
标识
DOI:10.1038/s41592-023-02028-1
摘要
Ribosome profiling has unveiled diverse regulation and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected messenger RNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with ordered two-template relay, a single-tube protocol for sequencing library preparation that incorporates adaptors by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling and termination. Our optimized methods for mRNA footprint generation and capture provide a richer translatome profile with low input and fewer technical challenges. A comprehensive redevelopment of the ribosome profiling workflow involves improved nuclease treatment and sequencing library preparation, enabling richer and more accurate translatome profiling with lower input and fewer technical hurdles.
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