Saccharomyces cerevisiae

Crm1, Nup1, and Nup2 bind upstream of and at the promoters of many genes in a manner that correlates with Pol II binding. To see if the loss of any one of these proteins can impact the transcriptome, we inhibited Crm1 or degraded Nup1/Nup2 and examined their effects on nascent and total RNA using thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq). Yeast cells were grown in synthetic complete medium containing glucose (SDC) before Crm1 was inhibited by treating the cultures with 100 ng/mL leptomycin B (LMB) in ethanol for 30 min at 30C. Nup1 and Nup2 were degraded by treating cultures with 500 uM indole-3-acetic acid (3-IAA) in ethanol for 16 hours at 30C. Following treatment, cultures were treated with 0.2 mM 4sU for 6 min at 30C. Cells were spun down and snap frozen. Total RNA was extracted using Phenol:Chloroform:IAA (25:24:1) extraction. 5 ug total RNA was treated with 10 mM iodoacetamide for 15 min at 50C. Libraries were made using the QuantSeq 3mRNA-seq Library Prep Kit for Illumina (FWD, Lexogen) following manufacturer recommendations. All experiments have at least three biological replicates.