Tbe gel

10 μL mixtures containing 1 μg purified DNA from MG1655 were incubated with 0–1.4 μg Histone H2A or LL-37 for 25 min at 25 °C. Gel loading sample buffer (5×, Bio-Rad, Hercules, CA) was added to a final concentration of 1× and the products were separated by native PAGE on a 5% TBE gel (Bio-Rad, Hercules, CA) at 100 V for 60 min. The gel was stained with 1X SYBR safe (Invitrogen, Carlsbad, CA) in TBE buffer⁷¹ for 30 min before visualization using a EOS Rebel T5 DSLR camera with an f/3.5–5.6 18–55 mm lens (Canon, Huntington, NY) and a DR46B Transilluminator (Clare Chemical, Dolores, CO).

For EMSA, ~150 nM pri-let-7 RNA was mixed with different MP^he proteins in a 1:1, 1:2 and 1:4 molar ratios in EMSA buffer-1 (25 mM HEPES pH 7.5, 100 mM NaCl, 2.5 mM DTT, 5 mM CaCl₂, 10 % glycerol, 0.01 % Triton X-100 and 1 U/ml RNasin). The reaction was incubated on ice for 1 hr, 20 % glycerol was added and analyzed on a 5 % TBE gel (Bio-Rad) run in ice-cold 0.5 X TBE buffer. Gels were stained with SybrGold and visualized on BioRad ChemiDoc imaging system.
For EMSA with SRSF3, 1 nM of the 5’ ³²P-labelled pri-miRNA was incubated with SRSF3 proteins at the indicated concentrations in EMSA buffer (25 mM HEPES pH 7.5, 75 mM NaCl, 2 mM DTT, 8% glycerol, 0.01 % Triton X-100, 1 mM EDTA, 0.1 mg/ml BSA) for 1 hr at 4°C. EMSA loading dye (50 % glycerol, 0.05 % xylene cyanol and 0.05 % bromophenol blue) was added and samples analyzed on a 20x20 cm 4 % TBE gel run in ice-cold 0.5 X TBE buffer. Gels were exposed to phosphor screens overnight and imaged using Typhoon FLA 7000 (GE healthcare Life Sciences).