5' RNA-Seq of yeast:rpb1-G1097D_VV448
This file was submitted without quality scores; placeholder quality scores present in the dumped files should be ignored. 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.