植物科学

Cucumber (Cucumis sativus) trichomes play a critical role in resisting external biological and abiotic stresses. Glandular trichomes are particularly significant as they serve as sites for the synthesis and secretion of secondary metabolites, while non-glandular trichomes are pivotal for determining the appearance quality of cucumbers. However, current methods for separating trichomes encounter challenges such as low efficiency and insufficient accuracy, limiting their applicability in multi-omics sequencing studies. This protocol introduces an efficient system designed for the precise separation of glandular and non-glandular trichomes from cucumber fruit. The process begins with the pre-cooling of sorbitol buffer or ethanol solution and the RNA-free treatment of laboratory supplies, followed by sterilization and pre-cooling. After filling glass bottles with pre-cooling buffer and glass beads, cucumber ovaries are then placed in the glass bottles and the trichome is harvested by bead-beating method. The separation process involves sequential filtration through various steel sieves and centrifugation to separate trichomes. The separated trichomes obtained from this method are well-suited for subsequent multi-omics sequencing analyses. This protocol achieved high precision in separating glandular and non-glandular trichomes, significantly enhancing the efficiency of separation and sample collection processes. This advancement not only addresses existing limitations but also facilitates comprehensive studies aimed at exploring the genetic and biochemical diversity present within cucumber trichomes, thereby opening avenues for broader agricultural and biological research applications.

Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from polyadenylated mRNA for transcriptome analysis. Both high-quality and high-throughput protocol options include steps of mRNA enrichment through magnetic bead-enabled precipitation of the poly-A tail, cDNA synthesis, and then fragmentation and adapter addition simultaneously through Tn5-mediated ‘tagmentation’. All steps of the protocols have been validated with Arabidopsis thaliana leaf and seedling tissues and streamlined to work together, with minimal cost in money and time, thus intended to provide a beginner-friendly start-to-finish RNA-seq library preparation for transcriptome analysis.

N⁶-methyladenosine (m⁶A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m⁶A can be detected by several methods, such as dot blot analysis using specific m⁶A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu et al., 2014; Shen et al., 2016). Here we describe the method for fast detection of total m⁶A levels in mRNA by dot blot analysis using a specific m⁶A antibody.

This protocol has been designed in order to facilitate the isolation and extraction of total RNA from microspores collected from lily anther sacs. This protocol allows the extraction of high amounts of high quality RNA, as observed in agarose gels.