Total RNA Extraction and Northern Blot

Each frozen cell pellet was resuspended in 240 μl TE (pH 8.0) plus 60 μl LETS buffer (50 mM Tris–HCl pH 8.0, 500 mM LiCl, 50 mM EDTA pH 8.0, 5% SDS) and 600 μl acidic phenol (pH∼3):chloroform (1:1). Lysates were sonicated in a fume hood, centrifuged, the aqueous phase extracted three more times with acidic phenol (pH∼3): chloroform and precipitated with ethanol. RNA was dissolved in water, treated with DNase (TURBO DNA-free Kit, Ambion) and visualized on a 7 M urea 7% polyacrylamide gel by staining with GelRed (Labmark).
RNAs were resolved on a 7% polyacrylamide gel and transferred onto an Amersham Hybond-N membrane according to the protocol described in Pánek et al. (2011) (link). 5′ biotinylated oligonucleotide probes were hybridized to the membrane and detected with BrightStar BioDetect Kit (Ambion) according to manufacturer’s instructions. For Northern blot probes sequences, see the Supplementary Material.