Single stranded dna oligos

Double-stranded DNA templates were designed to contain a 5’ T7 promoter to be used for in vitro transcription. Double-stranded DNA templates were prepared from single-stranded DNA oligos (Integrated DNA Technologies). DNA templates were amplified by PCR using Taq DNA polymerase (NEB M02373) and checked for quality using 1% agarose gel electrophoresis. PCR reactions were purified with the QIAquick PCR purification kit (Qiagen 28104).
In vitro transcription reactions using the AmpliScribe™ T7-Flash™ transcription kit (Lucigen ASF3507) were carried out at 37°0 for >4 h. Transcription reactions were terminated by treating with RNase-Free DNase I (Lucigen ASF3507) at 37°C for at least 15 min. RNAs were then purified from reactions by using Micro Bio-Spin Columns with Bio-Gel P-30 (Bio-Rad 7326223) followed by phenol-chloroform extraction with premixed acid phenol:chloroform:IAA (Invitrogen AM9732). Purified RNAs were then precipitated using isopropanol, and dissolved in nuclease-free water (Growcells UPW100012). Quality of samples was checked by running on a precast 6% TBE-Urea Gel (Life Technologies EC68655) at 180 V for 30 min. After staining with SYBR Gold (ThermoFisher S11494) diluted 1:10,000 in TBE buffer, RNA bands were imaged using a ChemiDoc MP (Bio-Rad) with a preset channel (ex 302 nm; em 590/110 nm).

Double-stranded DNA templates were designed to contain a 5’ T7 promoter to be used for in vitro transcription. Double-stranded DNA templates were prepared from single-stranded DNA oligos (Integrated DNA Technologies). Templates were amplified by PCR with Taq DNA polymerase (NEB M02373) and gel purified using QIAquick Gel Extraction Kit (Qiagen 28704).
In vitro transcription reactions were performed at 37°C for >10 h with the AmpliScribe™ T7-Flash™ transcription kit (Lucigen ASF3507). RNase-Free DNase I (Lucigen ASF3507) was used at 37°C for 15 min to terminate the reactions. RNA was purified from reactions using Zymo Research RNA Clean & Concentrator™-25 columns (Zymo Research R1707). The quality of RNA samples was checked with polyacrylamide gel electrophoresis using a precast 10% TBE-Urea Gel (Invitrogen EC62752BOX) at 250 V for 30 min. RNA bands were imaged using a ChemiDoc MP (Bio-Rad) with a preset channel (302 nm excitation and 590/110 nm emission) after staining with SYBR Gold (ThermoFisher S11494) diluted 1:10,000 in TBE buffer.

The 3’-FAM-labeled 30-mer RNA with 5’-end monophosphate [27 (link)] and the equivalent single-stranded DNA oligos were purchased from Integrated DNA Technologies (IDT). Exonuclease assays were performed at 37°C for 30 min with reaction mixtures containing 30 mM Tris (pH 8.0), 50 mM NH₄Cl, 2 mM MgCl₂, 0.5 mM dithiothreitol, 25 μg/ml bovine serum albumin, 2 μM 3’-FAM-labeled oligo, 0.1—1 μM DXO, and 0.01—0.1 μM Xrn1. The products were fractionated by 5% denaturing polyacrylamide gel electrophoresis and visualized on an ultraviolet illuminator. Assays were repeated at least three times to ensure reproducibility.