title: AT.rep3.rna
organism: Schizosaccharomyces pombe 972h-
description: Protocols: Cells were collected by filtration. Filters were immediately frozen in liquid nitrogen. Cells were grown at 32C in EMM2 (Moreno et al. Methods Enzymology 194: 795). Cells were grown in EMM2 (Moreno et al. Methods Enzymology 194: 795) in the presence of 10 mM 3-AT for 60 minutes at 32C. This treatment mimics histidine starvation. Total RNA was extracted using a hot phenol extraction method as described in Lyne et al. (2003) BMC Genomics 4:27, with an initial 45min incubation at 65 C. RNAs DNase treated, purified by Purelink micro columns and ribo-depleted with NEB rRNA depletion module (E7870L) with Yeast Probes 30 ng of ribo-depleted RNA was used as starting material for the NEXTflex Rapid Directional qRNA-Seq Kit V2 (Bioo Scientific).