RNA-seq of total and nascent RNA after depletion of Mpe1

To test the role of Mpe1 in transcription termination we inserted a mini-auxin induced degron (mAID) at the C-terminal end of the endogenous MPE1 locus in the yeast Saccharomyces cerevisiae. We treated Mpe1-mAID cells (JRY101) or wild type cells (WT, YMK728) with 1 mM auxin for 30 minutes and labeled the nascent RNA with 4-thiouracil (4tU) for 6 minutes. The nascent RNA fraction was prepared by biotinylating the 4tU-labeled RNA followed purification with streptavidin beads. Nascent and total RNA fractions were depleted of ribosomal RNA and strand-specific libraries prepared. Libraries were single-end sequenced using an Ilummina HiSeq 4000 instrument.