Brightstar biodetect kit protocol

Northern blotting was conducted as previously described (13 ). Briefly, 3 μg of isolated RNA was fractionated on 10% polyacrylamide gels containing 7% urea by electrophoresis at 55 V and subsequently, transferred to a Zeta-probe membrane (Bio-Rad) using a Trans-Blot SD semidry transfer apparatus (Bio-rad) at 4 mA per cm² with a maximum of 400 mA for 50 min. RNA was then UV-crosslinked to the membrane with a Spectroline UV crosslinker with the “optimal crosslink” setting. 5’-Biotinylated probes were hybridized to the membrane overnight at 42°C in ULTRAhyb (Ambion) hybridization buffer. Blots were developed according to the BrightStar BioDetect kit protocol (Ambion), imaged with the ChemiDoc MP imager (Bio-Rad), and individual band intensities were quantified using Image Lab software version 5.2.1 (Bio-Rad). Signal intensities for each transcript were normalized to that of 5S rRNA, which served as a loading control. Graphs of normalized abundance of each transcript for three biological replicates were produced using GraphPad Prism version 10.0.0.

RNA samples (2 μg) were loaded onto 10% Criterion Tris-buffered EDTA (TBE)–urea precast gels (Bio-Rad) and electrophoresed at 70 V until the dye front reached the bottom of the gel. RNA samples were transferred to a Zeta-Probe GT membrane (Bio-Rad) using a Trans-Blot SD semidry transfer apparatus (Bio-Rad) according to the manufacturer’s guidelines. Transferred RNA was UV cross-linked and hybridized overnight with 100 ng/ml of 5′-biotinylated probes (Table S2) in ULTRAhyb hybridization buffer (Ambion) at 42°C as described previously (29 (link), 30 (link)). Blots were developed using the BrightStar BioDetect kit protocol (Ambion) and imaged with a ChemiDoc MP imager (Bio-Rad). Quantifications of the bands were performed by densitometry tracing using Image J software.

Unless indicated otherwise, 2 μg of each RNA sample was fractionated on 10% Tris-Borate-EDTA (TBE) urea gels by electrophoresis at 55V. RNA was then transferred to a Zeta-probe membrane (Bio-Rad) using the Trans-Blot SD semidry transfer apparatus (Bio-Rad) per manufacturer’s instructions. RNA was UV-crosslinked to the membrane and probed for each given RNA using a 5′ biotinylated DNA oligo of complementary sequence in ULTRAhyb (Ambion) hybridization buffer at 42°C. Blots were developed using the Ambion Brightstar BioDetect kit protocol. Chemiluminescent signals were detected using the ChemiDoc MP imager (Bio-Rad) and image analysis was performed using the Image Lab software v. 6.0.1. Signal intensity for each sRNA was normalized to a control RNA (SsrA). RNA turnover curves and half-lives were generated using GraphPad Prism 8.