5' RNA-Seq of yeast:rpb1-E1103G_tfg2del146-180_VV520
Yeast strains were diluted from a saturated overnight YPD culture and grown to mid-log phase (~1.5x107/ml) YPD and harvested. Total RNA was extracted by a hot phenol-chloroform method (Schmitt et al., 1990), followed by on-column incubation with DNase I to remove DNA (RNeasy Mini kit, Qiagen), and processing with a Ribo-Zero rRNA removal kit (Epicentre) to deplete rRNA. To construct the cDNA library, samples were treated with terminator 5 phosphate-dependent exonuclease to remove RNAs with 5 monophosphate (5 P) ends, and remaining RNAs were purified purified and precipitated. Tobacco acid phosphatase (TAP, Epicentre) was added to convert 5 PPP or capped RNAs to 5 P RNAs. A SOLiD 5 adaptor was ligated to purified RNAs with 5 P (this step excludes 5 OH RNAs), followed by gel size selection of 5 adaptor ligated RNAs and reverse transcription (SuperScript III RT, Invitrogen) with 3 random priming. RNase H (Ambion) was added to remove the RNA strand of DNA-RNA duplexes, cDNA was size selected for 125-500 nt lengths.