GSM9652630: SHAPE-MaP of Ymu1 TnpB short reRNA scaffold; Escherichia coli; OTHER
For small RNA-seq: Purified reRNA was isolated directly from assembled TnpB RNP complexes using the RNA Clean & Concentrator-5 Kit (Zymo Research). Approximately 20 ng of purified RNA was end-repaired with QuickCIP (NEB, M0525S) and T4 PNK (NEB, M0201S). For SHAPE-MaP: 1 µg purified RNP was mixed with folding buffer (333 mM HEPES pH 8.0, 333 mM NaCl), combined with 10 mM MgCl2, and incubated at 37 °C. RNA was modified with 100 mM 1M7 in DMSO (modified) or DMSO alone (untreated control), then purified using the Qiagen RNeasy Micro Kit. For small RNA-seq: End-repaired RNA was used for library preparation with the Collibri Stranded RNA Library Prep Kit for Illumina Systems (Invitrogen, A38994024). Libraries were amplified with KAPA HiFi DNA Polymerase at 65 °C annealing temperature and size-selected by gel extraction on E-Gel EX 4% Agarose Gels. For SHAPE-MaP: Modified RNA was reverse-transcribed with SuperScript II using random primers and a transcript-specific primer in MaP buffer containing MnCl2 (25 °C 10 min, 42 °C 3 h, 70 °C 15 min). cDNA was purified, second-strand synthesis was performed with the NEBNext Second Strand Synthesis Module, and libraries were prepared with the Illumina Nextera XT kit (tagmentation at 55 °C, 14-cycle indexed PCR) and purified with AMPure XP beads (1.8×).