Ribolock rnase inhibitor

Sense and anti-sense DNA oligonucleotides for UBE3C-LRP5 fusion variants (v1, v2, and v7), LRP5, CTNNB1, LEF1, and scrambled siRNA (Supplementary Table S5) were ordered from Sigma-Aldrich. The protocol for the synthesis of small RNA transcripts using T7 RNA polymerase is reported in the literature^{69 (link)}. For each in vitro transcription (IVT) reaction, 1 nmol of each oligonucleotide (re-suspended in 1X TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA)) was annealed using thermocycler to obtain double-stranded DNA (dsDNA). The thermocycler conditions used were: 95 °C for 3 min, followed by 70 cycles of 95 °C for 30 s (−1 °C/cycle). In vitro transcription (IVT) reaction was performed in 20 µL of a reaction containing 1X T7 transcription buffer (cat. no. P118B, Promega), 1X biotin RNA labeling mix (cat. no. 11685597910, Sigma-Aldrich), 1 U RiboLock RNase Inhibitor (cat. no. EO0381, Fermentas), 10 U T7 RNA polymerase (cat. no. P2075, Promega), and 1 nmol of dsDNA, as a template. The reaction was incubated at 37 °C for 2 h. Sense and anti-sense small interfering RNA (siRNA) synthesized in separate reactions were annealed by mixing the transcription reactions at 95 °C for 1 min, followed by 70 cycles of 95 °C for 30 s (−1 °C/cycle) to obtain double-stranded siRNAs.