An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae
The precise regulation of transcription requires the coordinated activity of numerous proteins and protein complexes. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unresolved. Accurately defining the direct effects of transcriptional regulators is critical to addressing these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly synthesized RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly synthesized RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we assembled an end-to-end workflow to deplete proteins of interest using the AID system and measure newly synthesized RNA using SLAM-seq in the model eukaryote, Saccharomyces cerevisiae. We provide an open-source, step-by-step protocol to support rapid implementation of this workflow. We include methods for targeted protein degradation, 4-thiouracil (4tU) incorporation, rapid methanol fixation, RNA purification, RNA alkylation, mRNA-seq library construction, and data analysis. Additionally, we demonstrate that this workflow can help define the direct effects of transcriptional regulators using the bromodomain and extra-terminal domain (BET) proteins, Bdf1 and Bdf2, as an example. We provide evidence that data generated using this workflow is robust to normalization using whole-cell spike-in or total read counts, correlates well with 4tU-seq data, and identifies extensive differential expression due to the depletion of Bdf1 and Bdf2. Taken together, this workflow will help address outstanding questions underlying the molecular basis of transcriptional regulation and other processes in S. cerevisiae and other eukaryotes. Overall design: Saccharomyces cerevisiae cells were grown in YPD medium to an OD600 of ~0.45. Cultures were treated with 1 mM 3-indoleacetic acid (IAA) in DMSO for 25 min. Immediately after IAA treatment, cultures were treated with 5 mM 4-thiouracil (4tU) in DMSO for 4 min. Immediately after 4tU treatment, cultures were fixed in cold methanol. The final OD600 of the cultures was ~0.7. 4tU-labeled Schizosaccharomyces pombe cells were combined with S. cerevisiae cells in mass-to-mass ratio of 1:19 based on OD600. RNA was purified using a Quick-RNA Fungal/Bacterial Miniprep kit (Zymo Research) following the manufacturer's recommendations. Reagents were supplemented with DTT. Five µg of total RNA was treated with 10 mM iodoacetamide for 15 min at 50°C in the dark. 3' mRNA-seq libraries were constructed as described in the associated protocol at protocols.io. Libraries were sequenced on a NovaSeq 6000 (Illumina) at the OMRF Clinical Genomics Center. Demultiplexed, paired-end 150 bp reads were processed using fastp, bbduk, and SLAM-DUNK. All experiments were performed in biological triplicate.