Streptavidin conjugated magnetic beads

The N40 DNA aptamer library was purchased from TriLink BioTechnologies, Inc. (Ronkonkoma, NY). Other oligonucleotides, including amino-functionalized oligonucleotides, were synthesized by IDT (Coralville, IA), Sigma-Aldrich (St. Louis, MO), and Biosearch Technologies (Novato, CA). Carboxyl Dynabeads (14305D), the Oligreen single-stranded DNA (ssDNA) assay kit, and BODIPY fluorescent dye were purchased from Life Technologies, Inc. (Carlsbad, CA). Streptavidin-conjugated magnetic beads and λ exonuclease were purchased from New England Biolabs (Boston, MA). Azole drugs were purchased from Santa Cruz Biotechnologies (Dallas, TX). All other reagents and solvents were purchased from Thermo Fisher Scientific (Waltham, MA). Graphene devices were fabricated in house using methods described in previous work.

In vitro RNA pulldown assay was performed according to the previously reported method [51 (link)] with some modifications. Generally, RNA was heated in metal-free water for 2 min at 95 °C. The RNA was then flash-cooled on ice. The RNA 3X SHAPE buffer (333 mM HEPES, pH 8.0, 20 mM MgCl₂, 333 mM NaCl) was added and the RNA was allowed to equilibrate at 37 °C for 10 min. Then, 10 pmol of purified Flag-Elavl1a protein and 10 pmol of biotin-labeled probes were incubated with 15 μl streptavidin-conjugated magnetic beads (NEB) in binding buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.4 mM EDTA, 0.1% NP-40, 1 mM DTT, 0.4 U/μl RNase inhibitor) for 1 h at 4 °C. After washing with binding buffer for three times, the bead-bound proteins were heated in NuPAGE™ LDS Sample Buffer (4×) (Invitrogen) and then separated on the NuPAGE™ 4–12% Bis-Tris Gel (Invitrogen), and subjected to western blotting analysis with anti-Flag antibody (Sigma-Aldrich, F7425).

The rabbit polyclonal anti-Myc antibody and the biotin was purchased from Sigma-Aldrich. The mouse anti-EF1α antibody was purchased from Santa Cruz Biotechnology. The mouse monoclonal anti-TAC102 antibody and the rabbit anti-ATOM antibody have been described previously [24 (link),48 (link)]. Streptavidin-conjugated magnetic beads were purchased from NEB. HRP-conjugated streptavidin and AlexaFluor488-conjugated streptavidin were purchased from Thermo Fisher Scientific.