Rna loading dye

Ten nano meter 16 bp DNA (2 nM were labeled with ³³P at 5'-end) was digested with 0.66 nmol of enzyme at 37°C in 100 μl of a reaction buffer: 10 mM Tris-HCl, pH 7.5; 10 mM CaCl₂; 10 mM MgCl₂. 9 μl of the reaction mixtures were removed at 1, 2, 4, 8, 16, 32, 64, 128, 192 min after start. The samples were mixed with 9 μl of 2x RNA loading dye (Thermo Fisher Scientific, #SM1373), heated for 5 min at 95°C and analyzed by denaturing PAGE. The half-life of the substrate digestion was estimated during subsequent densitometry.

Toeprint assays were done according to (14 (link)). All steps were at 37°C unless otherwise specified. Before use, each mRNA together with dNTPs (0.5 mM final concentration) and radiolabeled ced70 primer (150 000 cpm) were denatured for 1 min at 90°C, followed by 1 min on ice. RNAs were refolded for 10 min in toeprint reaction buffer (10 mM Tris–HCl pH 7.6, 100 mM K-acetate, 10 mM Mg-acetate, 1 mM DTT). Pre-activated 30S (100 nM final concentration; 15 min at 37°C in 1× RT-buffer) were added for 10 min, followed by addition of tRNA^fMet (300 nM). 30S-IC formation was allowed for 20 min. Primer extension was carried out by addition of 50 U of SSIV RT (Invitrogen). After phenol/ chloroform/ isoamyl alcohol extraction (25/24/1), template RNAs were hydrolyzed by KOH treatment (300 mM) for 3 min at 90°C, followed by 1 h at 37°C. KOH was neutralized with acetic acid (600 mM), and the cDNA precipitated with Na-acetate and ethanol. After centrifugation and washes with 70% ethanol, cDNA was dissolved in 1 vol of water and 1 vol of 2× RNA loading dye (R0641, Thermo Fisher) and separated on an 8% sequencing gel. Signals were detected using a PhosphorImager screen and a PMI scanner™ (Biorad).

S.aureus Rnase P activity assays were performed as previously described [33 (link)]. Briefly, ptRNA^Tyr and rnpB RNA was denatured by heating to 95 °C for 5 min then slow cooled to room temperature. RNA species were then combined with 2× low salt buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl₂) and incubated for 5 min at 37 °C. Rnase P was reconstituted by mixing an equal molar ratio of His-RnpA and rnpB for 15 min at 37 °C. Precursor tRNA processing inhibition reactions were performed by combining 5 pmol of reconstituted Rnase P with DMSO (negative control) or 50 µM compound and incubating for 5 min at 37 °C. Then, 5 pmol ptRNA^Tyr was added to each reaction and incubated for an additional 30 min at 37 °C. Reactions were stopped with 2× RNA loading dye (Thermo Scientific) and heating to 65 °C for 10 min. Samples were electrophoresed on a 7M urea/8% polyacrylamide gel then stained with 0.5 µg mL⁻¹ ethidium bromide. The BioRad EZ Gel Doc imaging system was used to visualize the RNA and relative abundance of mature tRNA^Tyr in the DMSO control or in the samples containing compound and analyzed using BioRad Image Lab densitometry software. The percent inhibitory activity of each compound was calculated using the following equation: (% experimental processing/% processing negative control) × 100.

Total RNA was extracted from tumor tissue by using Trizol Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. RNA quality was confirmed by electrophoresis on a 1% agarose gel in 2× RNA loading dye (Thermo Fisher Scientific). For genomic DNA removal, the RapidOut DNA Removal Kit (Thermo Fisher Scientific) was used. For cDNA synthesis, the SuperScript II Reverse Transcription Kit (Thermo Fisher Scientific) with random hexamers (Microsynth, Balgach, Switzerland) was employed. Real‐time quantitative polymerase chain reaction (RT‐qPCR) analysis was performed on a Bio‐Rad CFX96 (Hercules, CA) using the Brilliant III Ultra‐Fast Quantitative PCR Kit (Agilent technologies, Santa Clara, CA) and TaqMan probes. Probes and primers were purchased from Thermo Fisher Scientific (CCL2 Mm00441242_m1, CCL3 Mm00441259_g1, CCL4 Mm00443111_m1, CXCL9 Mm00434946_m1, CXCL10 Mm00445235_m1, Galectin‐9 (Lgals9) Mm00495295_m1, Gp100 Mm00498996_m1, H60b Mm04243254_m1, H60c Mm04243526_m1, IL‐10 Mm00439614_m1, IL‐12a Mm00434165_m1, IL‐12b Mm01288989_m1, IL‐15 Mm00434210_m1, IL‐18 Mm00434225_m1, PD‐L1 Mm00452054_m1, PD‐L2 Mm00451734_m1, Rae1 Mm00558293_g1, TGF‐β1 Mm01178820_m1, Trp‐2 Mm01225584_m1, Ulbp1 Mm01180648_m1). Sequences for probes and primers specific for TATA binding protein (Tbp) were synthesized by Microsynth.