GSM8084405: Arid5a, 0.25 µM; synthetic construct; RNA-Seq
For the RBNS experiment, a random 20-mer RNA input pool generated by in vitro transcription was used. For this as a template, a T7 promoter sequence and a random 20-mer containing oligonucleotide was annealed to an equimolar amount of a shorter oligonucleotide containing only the complementary T7 promoter sequence. The resulting RNA was folded by snap cooling prior to use. Adapted from https://doi.org/10.1016/bs.mie.2015.02.007, an RBNS assay was performed using the random 20-mer RNA input pool and a Twin-Strep-labelled Arid5a49-152 domain. The protein, equilibrated for 30 min at 4°C 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), was incubated with the input RNA pool (1 µM) and 40 U of ribonuclease inhibitor (moloX, Berlin). This was done for 1 h at room temperature at three different protein concentrations (0.25, 1, 5 µM). The final pull-down was performed using MagStrep "type3" XT beads (IBA Lifesciences, Göttingen) by incubating the RNA/RBP mixture for 1 h at 4 °C. After three washing steps with wash buffer (25 mM Tris-HCl pH 8.0, 150 mM KCl, 60 μg/ml BSA, 0.5 mM EDTA, 0.01% Tween), 25 µL of elution buffer (wash buffer plus 50 mM biotin) was used twice to elute the RNA/RBP complexes from the beads. 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).