Maxiscript t7 transcription kit

G4-forming PIM1 and control ΔG4 sequences were taken from ^{48 (link)}. Two additional control RNAs, one for which the Gs within the G4-forming sequence were mutated to non-Gs (G-to-H) and a second for which the Gs within the G4-forming sequence were mutated to non-Gs and an equal number of non-G nucleotides outside of the G4-forming sequence were mutated to Gs (G-rich), were synthesized as gBlocks (IDT, sequences in Supplementary Table 2) and cloned into pcDNA3.1. Linearized vectors were transcribed using the MAXIscript T7 Transcription Kit (Thermo Fisher Scientific) and RNA treated with Turbo DNase (Thermo Fisher Scientific). Biotin-14-CTP (19519016 Life Technologies) was added in a 0.4:1 ratio relative to CTP. RNA integrity was verified by polyacrylamide gel electrophoresis. G4 structure formation was confirmed using a reverse transcriptase stalling assay ^{68 (link)}. [rG₄rA₄]₅, 5’-biotinylated-[rG₄rA₄]₅, [rGrA]₂₀ and 5’-biotinylated-[rGrA]₂₀ 40-mer RNA oligonucleotides were obtained from IDT. Native gel electrophoresis to measure formation of secondary structure was performed as described ^{35 (link)}. RNA was folded either as described ^{35 (link)} or in pull-down buffer to confirm maintenance of RNA structure during PRC2 pull-down assays. Radiolabeled RNA was visualised using a Typhoon phosphorimager (GE) and ImageQuantTL (GE).

Matrices for in vitro transcription were amplified from the plasmids pBAD2-bgaB containing cspA 5′ UTR and its mutated forms with primers cspA_T7_F and cspA_T7_R (Table S6). In vitro transcription was performed with MAXIscript T7 Transcription Kit (Thermo Fisher Scientific) and the synthesized RNA was purified on 6% denaturing polyacrylamide gel (19:1 acrylamide/bis-acrylamide ratio). To study the effect of temperature on folding, 50 ng of cspA 5′ UTR was dissolved in 9 μL of Loading buffer (10 mM Tris-HCl pH7.0, 1 mM EDTA, 10% glycerol, 0.01% xylene cyanol), denatured at 95°C for 3 minutes and quickly cooled on ice. To initiate refolding, 1 μl of 500 mM NaCl solution was added, and RNA was incubated at temperatures ranging from 26°C to 37°C for 5 minutes. The folded RNA was applied to the 10% acrylamide gel containing 100 mM Tris-HEPES (pH = 7.5), 0.1 mM EDTA, 3 mM MgCl₂, and run at 4°C with a running buffer of the same composition. The RNA fragments in the gel were visualized by staining with SYBR Gold stain (Thermo Fisher Scientific).