GSM7596284: RBNS, RHAU53, Synthesized construct, 5nM, Li+; synthetic construct; OTHER
RNAs with a random region of 40nt were prepared by in vitro transcription (NEB, E2040S) and incubated with an increasing concentration of RHAU53 (5, 20, 80, 320 and 1300nM). The RNA pool resolved by 10% PAGE gel and purified (Zymo Research, R1016). Concentrated RNAs were heated at 85C for 5 min in the presence of 150 mM KCl or LiCl and allowed to cool for at least 3 min to allow for folding. 10 µl NEBExpress® Ni-NTA Magnetic Bead (NEB, S1423L) was used to replace the Dynabeads MyOne Streptavidin T1 in old method to pull-down the His tagged protein-RNA complexes. All wash and binding buffers contained either 150 mM KCl or LiCl. Protein-RNA complexes were eluted in the final buffer concentration of 20 mM Tris-HCl (pH 7.5), 150mM KCl or LiCl, 500mM Imidazole (pH 7.4) and 8M Urea. RNAs were isolated and reversely transcribed for PCR amplification and sequencing RNA Bind-n-Seq