(A) Four in vitro synthesized and adenylated transcripts with the indicated average poly(A)-tail length were spiked into a fixed amount of total HeLa RNA (800 ng) and analyzed by ePAT and LM-PAT (see Supplemental Material for details of in vitro transcripts). (B) Schematic representation of the time course of galactose induction and glucose repression. The arrows indicate the time points at which cells were harvested. (C) The 10-min time point represents newly synthesized GAL1 transcript with a long poly(A)-tail. The GAL1-(long) and GAL1-(short) transcripts are generated by alternate poly(A) site usage. (D) The time-dependent shortening of the two GAL1 amplicons was quantified relative to the 100-bp ladder and the migration of the TVN product. This is only really possible using the ePAT assay, since laddering in the LM-PAT assay results in two peaks of similar intensity at the last time point of this assay (20 min).
