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===Attach RNA to Streptavidin Beads=== *[https://www.thermofisher.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/protein-isolation-protocol/dynabeads-myone-streptavidin-c1.html|Dynabeads MyOne C1 protocol] *Make Solutions A & B and Binding&Wash Buffer **Solution A (DEPC-treated 0.1 M NaOH, DEPC-treated 0.05 M NaCl): 2.5ml 1M NaOH + 0.25ml 5M NaCl + 22.25ml nf-H2O ***NaOH: 39.997g/mol -> 1M NaOH = 0.1g NaOH dissolved in 2.5mL nf H2O **Solution B (DEPC-treated 0.1 M NaCl): 0.5ml 5M NaCl + 24.5ml nf-H2O **2X B&W Buffer (10 mM Tris-HCl 1 mM EDTA 2M NaCl): 10ml 5M NaCl + 50ul 0.5M EDTA + 62.5ul 4M Tris-HCl + 14.8875ml H2O #Resuspend beads by vortexing 30sec #Transfer 100ul of beads (10ug/ul) to new tube #Add 1ml 1X B&W Buffer and mix #Pull down by magnet 2min and remove supernatant #Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant #Wash 2x with 100ul Solution A for 2 min each #Wash 2x with 100ul Solution B #Resuspend the beads in 200ul 2X B&W Buffer #Add 200ul biotinylated RNA #Incubate 15min at RT gently rotating #Pull down with magnet for 3min #Wash 3x with 1X B&W Buffer #Resuspend in 60ul nf H2O
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