Vacuum gel drier

For EMSAs with short RNAs (<100 nt), protein at the indicated final concentration was mixed with 5 nM radiolabeled RNA in RNase-free protein buffer supplemented with 4% glycerol and 30 µg per mL yeast tRNA as a competitor in a final volume of 20 µL. In order to allow protein–RNA complexes to form, the mixtures were incubated for >20 min at RT.
Separation of protein–RNA complexes was performed by native PAGE on 6% polyacrylamide 1x TBE gels in 40 min at constant 110 V in 1x TBE running buffer. Subsequently, the gels were fixed in 30% (v/v) methanol, 10% (v/v) acetic acid for 10 min before drying in a vacuum gel drier (BioRad). Visualization of radioactivity occurred after exposure of radiograph films (Kodak) in a Protec Optimax developer (Hohmann) or by PhosphorImaging with a Fujifilm FLA-3000. Each experiment was performed as a triplicate on different days.
In the case of long, unlabeled RNA (>100 nt), 10–100 nM RNAs were used, and separation of protein–RNA complexes was performed by 1–1.5% agarose gel electrophoresis. Visualization of RNA was achieved by GelRed (Biotium) staining. Fluorescence was visualized with a Fusion SL imaging system (Vilber Lourmat) by UV at 254 nm.

Telomere overhang analysis was performed as described in our previous manuscript (Shastrula et al., 2018 (link)). In summary, pure genomic DNA was isolated from cells using a GeneJET purification kit (Thermo Fisher Scientific). 15 μg was then digested with ExoI (New England Biolabs) overnight. The next day, AluI and MboI (New England Biolabs) were added to the reaction, and 15 μg of control (non–ExoI treated) DNA was also digested. DNA was then purified through ethanol precipitation and run on a 0.7% large agarose gel. The gel was dried using a vacuum gel drier (Bio-Rad) for 30 min, and the dried gel was hybridized with a ³²P-labeled (5′-CCCTAA-3′)4 probe in Church buffer overnight and at 42°C. The gel was washed twice with 0.25× SSC (a ready-to-use saline sodium citrate buffer, pH 7.0–7.5, that contains 0.3% NP40 and Proclin 950 as a preservative) with 0.1% SDS and then 0.25× SSC for 30 min each. The gel was exposed to a phosphor-imager (GE Healthcare) and visualized with a Typhoon RGB Imager (GE Healthcare). To reprobe the gel for total telomeric signal, the gel was denatured in alkaline solution (0.5 M NaOH and 0.15 M NaCl) twice for 30 min each and neutralized in 0.5 M Tris-HCl, pH 7.5, and 3 M NaCl twice for 20 min each. The gel was preequilibrated in 5× SSC for 15 min and hybridized as above with a ³²P-labeled (5′-CCCTAA-3′)4 probe.