Yong Zhang, Ye Xu, T. Skaggs, J. F. Ferreira, Xuemei Chen, D. Sandhu
{"title":"An advanced protocol for profiling RNA-binding proteins in Arabidopsis using plant phase extraction","authors":"Yong Zhang, Ye Xu, T. Skaggs, J. F. Ferreira, Xuemei Chen, D. Sandhu","doi":"10.1093/biomethods/bpad016","DOIUrl":"https://doi.org/10.1093/biomethods/bpad016","url":null,"abstract":"\u0000 RNA-binding proteins (RBPs) are key players in regulating cell fate and essential developmental processes. Systematic profiling of the RNA-binding proteome (RBPome) is thus indispensable for researchers aiming to understand the mechanisms of post-transcriptional gene regulation. RBPome identification methods developed in humans, mice, and bacteria have successfully identified RBPomes in these organisms. However, the biochemical and genetic complexities of plant tissues have greatly hindered the effectiveness of these methods in plants. Moreover, plant RBPs have been predominantly discovered through oligo d(T) based affinity purification (RNA-interactome capture, RIC). Since polyadenylated RNA only accounts for less than 5% of the total RNA population in eukaryotic cells, there is a pressing need to develop a comprehensive yet unbiased method to capture the full spectrum of RBPs in plants. Here, we describe a detailed protocol of Plant Phase Extraction (PPE), a recently developed method to identify RBPs in Arabidopsis [1]. The PPE method enables the efficient enrichment of both poly(A) and non-poly(A) RBPs from various tissues quickly and reproducibly. Most importantly, PPE allows for unveiling dynamic RBP-RNA interactions under various abiotic and biotic stress conditions and during different plant developmental stages. This provides a much broader and more accurate understanding of plant RBPs, marking a significant advancement in plant molecular biology.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"1 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44031174","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Protocol for transcriptome assembly by the TransBorrow algorithm","authors":"Dengyi Zhao, Juntao Liu, Ting Yu","doi":"10.1093/biomethods/bpad028","DOIUrl":"https://doi.org/10.1093/biomethods/bpad028","url":null,"abstract":"Abstract High-throughput RNA-seq enables comprehensive analysis of the transcriptome for various purposes. However, this technology generally generates massive amounts of sequencing reads with a shorter read length. Consequently, fast, accurate, and flexible tools are needed for assembling raw RNA-seq data into full-length transcripts and quantifying their expression levels. In this protocol, we report TransBorrow, a novel transcriptome assembly software specifically designed for short RNA-seq reads. TransBorrow is employed in conjunction with a splice-aware alignment tool (e.g. Hisat2 and Star) and some other transcriptome assembly tools (e.g. StringTie, Cufflinks, and Scallop). The protocol encompasses all necessary steps, starting from downloading and processing raw sequencing data to assembling the full-length transcripts and quantifying their expressed abundances. The execution time of the protocol may vary depending on the sizes of processed datasets and computational platforms.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"23 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135447898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}