NaP-TRAP: A versatile and accessible workflow to dissect principles of translational regulation and mRNA stability

regulatory impact of elements distributed throughout the mRNA, from cap-to-tail. This protocol covers experimental design, reporter construction, sample processing, and computational analysis for both low- and high-throughput applications. Bench work can be completed in 4-5 days, with qPCR-based readouts requiring only basic Excel skills for data processing. Sequencing-based readouts require skills in command-line tools and Python scripting and add an additional 2-3 days. NaP-TRAP thus offers an accessible, robust, and quantitative platform to decode the regulatory logic of mRNA translation and stability in diverse biological contexts. Basic Protocol 1: Design, assembly, and synthesis of NaP-TRAP reporter libraries. Support Protocol 1: Design, assembly, and synthesis of NaP-TRAP individual reporters and spike-ins. Basic Protocol 2: NaP-TRAP delivery by micro-injection in zebrafish embryos. Alternate Protocol 1: NaP-TRAP delivery by transfection in cultured mammalian cells. Basic Protocol 3: NaP-TRAP pulldown and RNA extraction. Basic Protocol 4: Preparation of NaP-TRAP cDNA Sequencing Libraries. Alternate Protocol 2: NaP-TRAP-qPCR module for low-cost validation. Basic Protocol 5: Computational analysis of NaP-TRAP MPRA data.