Small RNA-Seq of homo sapiens:8-tandem targeting group-2
The Small RNA-seq experiments were performed as followed. Cells were co-transfected with 4 g of plasmid DNA expressing an 8-tandem tigRNA array and TIGRa-NFZ at a 1:1 molar ratio, using polyetherimide (PEI; YEASEN) at a DNA:PEI ratio of 1:2 in OptiMEM (Gibco) according to the manufacturer`s protocol. At 24 h post-transfection, the culture medium was replaced with medium containing 2 g/mL puromycin for selection. Total RNA was extracted 5 days after transfection using TRIzol Reagent (Invitrogen) and submitted for small RNA sequencing (Azenta Life Sciences). Raw sequencing reads were processed with fastp (v0.24.0; parameters: --low_complexity_filter -a AGATCGGAAGAGCACACGTCT) and aligned to the tigRNA reference sequence using STAR (v2.7.11b). Alignments were filtered to retain only reads 15 nt in length that showed perfect matches across all aligned regions (i.e., without insertions, deletions, or soft-clipping). Processed alignment files were then converted using bamCoverage (v3.5.6) and visualized with Python (v3.10.19).