RNA-RNA and RNA-protein interactions are involved in the regulation of gene expression. Here, we describe an updated and extended version of our RNA purification and protein identification (RaPID) protocol for the pulldown of aptamer-tagged mRNAs by affinity purification. The method takes advantage of the high affinity interaction between the MS2 RNA aptamer and the MS2 coat protein (MCP), as well as that between streptavidin-binding peptide (SBP) and streptavidin. Thus, it employs MCP-SBP fusions to affinity purify MS2-tagged target RNAs of interest over immobilized streptavidin. Purified aptamer-tagged mRNAs, along with any associated RNAs and proteins, are then sent for RNA sequencing (RaPID-seq) or mass spectrometry (RaPID-MS), which allows for the identification of bound cohort RNAs and proteins, respectively.

Transcription and RNA decay play critical roles in the process of gene expression and the ability to accurately measure cellular mRNA levels is essential for understanding this regulation. Here, we describe a single-molecule fluorescent in situ hybridization (smFISH) method (as performed in Haimovich et al., 2017) that detects single RNA molecules in individual cells. This technique employs multiple single-stranded, fluorescent labeled, short DNA probes that hybridize to target RNAs in fixed cells, allowing for both the quantification and localization of cytoplasmic and nuclear RNAs at the single-cell level and single-molecule resolution. Analyzing smFISH data provides absolute quantitative data of the number of cytoplasmic (“mature”) mRNAs, the number of nascent RNA molecules at distinct transcription sites, and the spatial localization of these RNAs in the cytoplasm and/or nucleoplasm.

Plasmids are widely used tools in yeast research. In many cases, plasmid libraries are used in genetic screens or in yeast two hybrid screens. In such cases, it is necessary to extract plasmids carrying unknown genetic elements from positive clones that were isolated in the screen.

This is a simple protocol to extract plasmid DNA from budding yeast cultures (Robzyk and Kassir, 1992). The amount produced is small, but it is sufficient for PCR or for transformation into bacteria, where the plasmid can be amplified to provide sufficient amounts for downstream uses (e.g., restriction enzyme analysis, sequencing).