title: GSM8353403: CDE N(3-5), 500 nM; synthetic construct; OTHER
library_name: GSM8353403
library_construction_protocol: For the sRBNS experiment, a Twin-Strep tagged coreROQ domain and different pre-structured RNA input pools were used. The three different RNA input pools [CDE pool N(3), CDE pool N(3-5), ADE pool N(6)] were generated by in vitro transcription. For the sRBNS experiment, we adapted the protocol from https://doi.org/10.1016/bs.mie.2015.02.007. The protein was equilibrated in binding buffer (25 mM Tris-HCl pH 7.5, 150 mM KCl, 3 mM MgCl2, 0.01% Tween, 500 µg/mL BSA, 1 mM DTT) at three different concentrations (10, 50, 500 nM) for 30 min at 4°C. Second, the folded RNA was added to a final concentration of 1 µM together with 40 U of ribonuclease inhibitor (moloX, Berlin) and incubated for 1 h at room temperature. Pulldown was performed by incubating the RNA pool/coreROQ mixture with 1 μL of washed MagStrep "type3" XT beads (IBA Lifesciences, Göttingen, Germany) for 1 h at 4°C. Unbound RNA was then removed by washing three times with wash buffer (25 mM Tris-HCl pH 8.0, 150 mM KCl, 60 μg/ml BSA, 0.5 mM EDTA, 0.01% Tween). The RNA/coreROQ complexes were then eluted twice with 25 µL of Elution Buffer (wash buffer containing 50 mM biotin). RNA was then extracted using the Zymo RNA Clean & Concentrator-5 Kit (Zymo Research, Freiburg, Germany) according to the manufacturer's instructions. The RNA extracted after pulldown was transcribed into cDNA. The cDNA was then amplified by PCR, using primers to insert an index for each concentration and Illumina adapters. Deep sequencing was then performed at GENEWIZ (Leipzig, Germany).