Urea page gels

Primer extension reactions were performed using a modified T7 DNA polymerase (Sequenase™ version 2.0, Thermo Fisher) and a Cy5-labelled primer (P3: 5′-Cy5-TAATGTGAGTTAGCT-3′) annealing to the 3098-nt ssDNA templates and 230 nt from the start of the STRs or designed structures. Each reaction contained 0.5 μL of 10 μM fluorescently labelled P3, 2.5 μL of 100 ng/μL of the ssDNA templates in 7 μL of the reaction buffer. The final composition of the reaction buffer was 40 mM Tris.HCl pH 7.5, 20 mM MgCl₂, 50 mM NaCl and 50 mM KCl. The mix was incubated for 2 min at 80 °C and slowly cooled down to 20 °C over 1 h; 1 μL of 0.1 M DTT, 1.5 μL of 10 mM dNTPs and T7 DNA polymerase (1.625 units) were then added for a total volume of 15 μL. The reactions were carried out at room temperature. At the indicated time points, the primer extension reactions were stopped by adding a formamide loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol), and the products were separated on a 6% urea-PAGE gels (Invitrogen) run at 180 V for 50 min. To assess the position of the stalled products, the assay was performed using two templates containing either a G4 motif (GGGAGGGTGGGAGGG) or a mutated form of the same sequence (GGGACCCTGGGAGGG) not expected to support G4 formation.

The templates for in vitro transcription reactions were either amplified by PCR (SEC61A1, TMEM109, TMED7, DDOST) or by linearizing plasmids (ATPV0B, BLOC1S1). After gel purification, the linearized plasmid templates were cleaned up and concentrated to 500 ng/µL with Zymo Clean-UP and concentrator 5 columns. RNA was transcribed using HiScribe high yield T7 kit (New England Biolabs) for 2 hr at 37°C. The reactions were stopped by addition of DNaseI and incubation for 15 min at 37°C. The RNA products were PAGE separated using 6% urea-PAGE gels (Invitrogen). The RNAs were excised from the gels with a razor blade and the gel fragments crushed into small pieces. The RNAs were extracted from the gels using 3 gel volumes of RNA extraction buffer (300 mM NaOAc, 1 mM EDTA, and RNasin prepared in RNAse-free water). For extraction, the gel slurry was incubated at 20°C shaking for an hour. The slurry was transfered to a Spin-X 0.45 μm tube filter and spun at 15,000 rpm for 3 min to filter out the gel pieces. The RNAs were precipitated from the filtrate with isopropanol, air dried and resuspended in RNase-free water. The transcribed RNAs were then folded by incubating them for 5 min at 95°C and cooling down to 25°C at 1 °C/min in a thermocycler.