RNA Extraction and Northern Blot Analysis

For most experiments, cells were cultured in YEA medium that contains phosphate. In experiments comparing levels of pho1 expression in the presence or absence of phosphate, EMM was used with or without 15.5 mM sodium phosphate and 20 mM potassium phosphate. Total RNA was isolated by incubating cells in hot phenol heated to 65 °C for 10 min followed by 3 additional extractions using phenol-chloroform. RNA was precipitated using the sodium-acetate-ethanol method. Northern blots were performed according to the published protocol^{6 (link)}. 10 µg of RNA was resolved on a 1% formaldehyde-agarose denaturing gel and capillary transferred using NorthernMAX transfer buffer (Thermo Fisher Scientific) onto positively charged BrightStar-Plus nylon membrane (Ambion) and crosslinked using UV Stratalinker 2400 (Stratagene). The T7 in vitro transcription kit (Promega) was used to generate α-P³²-UTP (PerkinElmer) labeled RNA probes (Supplementary Table 2) that were hybridized to the membrane overnight at 65 °C in ULTRAhyb buffer (Ambion). The membrane was exposed and scanned using a Typhoon FLA 9500 phosphor imager (GE Healthcare).