T7 flash biotin rna transcription kit

The DNA template used for biotinylated circATXN7 in vitro synthesis was generated by PCR and purified using a DNA Gel Extraction Kit (Axygen). In vitro transcription was performed using the T7-Flash BiotinRNA Transcription Kit (Epicentre, biotin labeling) according to the manufacturer’s instructions. RNA was subsequently purified by phenol-chloroform extraction. For linear RNA in vitro cyclization, the RNA products were incubated with the indicated DNA splints (molar ratio = 1:1.5) at 90 °C for 5 min, and then cooled to room temperature over 20 min. Ligation to form circRNAs was then performed overnight at 16 °C with T4 DNA ligase (NEB). The sample was then treated with RNase R and DNase I at 37 °C for 30 min and subsequently purified by phenol-chloroform extraction.

The DNA template used for the in vitro synthesis of biotinylated H19 was amplified by PCR. The forward primer contains the promoter sequence for T7 RNA polymerase, enabling further in vitro transcription. PCR products were purified with the DNA Gel Extraction Kit (AxyPrep, Corning, NY, USA) according to the manufacturer’s instructions and in vitro transcription was conducted with the T7-Flash Biotin RNA Transcription Kit (Epicentre, Madison, WI, USA).

pcDNA3 vectors harbouring full length or mutant REG1CP respectively were used as templates for in vitro transcription of biotin-labelled RNAs. The forward primer contained the T7 RNA polymerase promoter sequence to allow for subsequent in vitro transcription. PCR products were purified using DNA Gel Extraction kit (Bioline; BIO-52058) and in vitro transcription was performed using T7-Flash Biotin-RNA Transcription Kit (Epicentre) according to the manufacturer’s instructions.

DNA templates incorporating the T7 RNA polymerase promoter sequence were generated by PCR amplification and then purified using the DNA Gel Extraction Kit (Thermo Fisher). In vitro transcription was performed using the T7‐Flash Biotin‐RNA Transcription Kit (Epicentre) according to the manufacturer's instructions. Biotin RNA pull‐down assays was performed as previously described.^[27, ^32] Briefly, 1–2 × 10⁷ cells were lysed in 1.0 mL of RIP buffer (50 mm Tris‐HCl pH 7.5, 150 mm NaCl, 2.5 mm MgCl2, 1 mm EDTA, 5% glycerol, 0.5% NP‐40, 1 mm DTT, supplemented with Protease Inhibitor Cocktail (Roche), 40 U mL⁻¹ RNase inhibitor, and 20 µm MG132). Cell lysates were then incubated with streptavidin beads (Invitrogen) coated with the biotin‐labeled RNA or DNA probes (3 µg) at 4 °C for 4 h overnight. Beads were washed five times in RIP buffer (without glycerol) and eluted in Laemmli buffer. The samples were separated by SDS‐PAGE prior to mass spectrometry or western blotting analysis.