Rnase if

EVs containing miR-424 were isolated from RWPE-1 miR424 stable cells and incubated with RNase If (1 µL, 50,000 units/mL, NEB, Ipswich, MA) for 20 min at 30 °C, or with the addition of 1% Triton. Following incubation, RNA was extracted using Direct-zol Miniprep kit as described above.

Cleavage efficiency was measured by a dose–response of RNP against 2 nM DNA substrate for 1 h at 37°C. RNP was added and mixed with DNA substrate using a Biomek FX^p liquid handler. Activity was quenched by the addition of Proteinase K followed by RNase If (NEB). Fraction of cleaved substrate was measured using the Fragment Analyzer automated CE system (AATI). Analysis was performed with GraphPad Prism using a four-parameter dose-response curve.

Complementary DNA (cDNA) from rRNA-depleted RNA was prepared using the Superscript III cDNA synthesis kit (Invitrogen). In brief, 200 ng of RNA was resuspended in 17 μl nuclease-free water and heated at 95 °C for 5 min to fragment the RNA and thus improve the eventual evenness of coverage. Random hexamers (10 μl of 50 ng/ul) and dNTPs (2 μl of 10 mM mix) were added to the RNA and the mixture was heated to 65 °C for 5 min, and then placed on ice for 2 min. A mixture of 10X buffer (5 μl), 0.1 M DTT (1 μl) and 25 mM MgCl2 (10 ul) was added to the tube and the mixture was incubated at 15 °C for 20 min. A mixture of RnaseOut (2.5 ul) and SSIII reverse transcriptase (2.5 μl) was then added to the tube and the sample incubated (25 °C 10 min, 40 °C 40 min, 55 °C 50 min, 85 °C 5 min, 4 °C hold). The RNA remaining in the sample was then degraded by the addition of 1 μl RNase H and 1 μl RNase If (New England Biolabs, M0243L). The resulting cDNA was then purified by the serial use of two Performa Gel Filtration Columns (EdgeBio, 42,453). The concentration of the resulting cDNA was then quantified using the Nanodrop as previously described [51 (link)].

Nonselective YPDA and YPG yeast growth media and synthetic media with drop-out mix were prepared as described (Sherman 2002 (link)). For selection of resistant strains, G418 sulfate and hygromycin B were added to media at concentrations of 200 and 250 µg/ml, respectively. Escherichia coli cells were grown in LB or TB broth-based media (Green and Sambrook 2012 ). The high-efficiency lithium acetate method described by Gietz and Schiestl was used for DNA transformations involving gene disruption (Gietz and Schiestl 2007 (link)) and a rapid lithium acetate/DMSO transformation method was used for simple plasmid transformations (Tripp et al. 2013 (link)). Yeast chromosomal DNA was purified as described (Lee et al. 2012 (link)). Plasmid DNAs were purified using Qiagen Qiaprep kits. PCR reactions utilized an Applied Biosystems 2720 Thermal Cycler. Restriction enzymes, Antarctic phosphatase, RNase I_f, and Phusion high-fidelity DNA polymerase were obtained from New England Biolabs. The chemical 5-fluoroorotic acid (5-FOA) was purchased from Gold Biotechnology.

TECs were purified as described above and split into 25-μl aliquots from a common pool. RNA degradation was performed by adding 0.5 μl of RNase I_f (NEB) alone or in combination with 0.5 μl of RNase Cocktail (Thermo Fisher Scientific), which contains RNase A and RNase T1, and incubating at 37 °C for 15 min. The untreated control sample was kept at RT during this time. 15 μl of each sample was gently mixed with 3-μl 6X native DNA loading dye and assessed by EMSA as described below in the section Analysis of transcription complexes by EMSA.