Journal: bioRxiv
Article Title: NaP-TRAP: A versatile and accessible workflow to dissect principles of translational regulation and mRNA stability
doi: 10.64898/2026.04.12.718002
Figure Lengend Snippet: NaP-TRAP reporter assembly and sequencing library preparation. ( A ) Complex NaP-TRAP reporter libraries for MPRA applications are generated by PCR-based assembly, in which pooled inserts are fused to the shared FLAG–GFP reporter backbone and flanking sequences required for in vitro transcription. ( B ) Single reporters can be cloned individually and linear in vitro transcription templates produced by PCR using primers that add the appropriate promoter and hard-encoded poly(A) tail. ( C ) For qPCR readout, reporter abundance in matched Input and Pulldown fractions is quantified using insert-specific primer sets and normalized to a co-delivered control reporter (e.g., dsRed). ( D ) Example design of a tiled 5′-UTR library, in which endogenous sequences are sampled using 124-nt windows with 25-nt steps. ( E ) For sequencing readout, NaP-TRAP RNA is reverse transcribed using a custom barcoded RT primer that incorporates a sample barcode, a 10N unique molecular identifier (UMI), and an i7 index sequence. Subsequent PCR-amplification with indexed Illumina primers yields a dual-indexed sequencing library compatible with Illumina platforms. ( F ) Reverse-transcribed samples with distinct custom barcoded RT primers can be pooled prior to the final PCR amplification with indexed Illumina primers. This pooling step promotes uniform PCR amplification across replicates. After sequencing, reads from each replicate are recovered during demultiplexing using the RT primer barcode.
Article Snippet: Superscript III enzyme (Thermo Fisher Scientific, Cat. No. 18080044) RNase H (New England Biolabs, Cat. No. M0297S) RNase If (New England Biolabs, Cat. No. M0243S) D1000 Screen Tape and Sample Buffer (Agilent, Cat. No. 5067-5582, 5067-5583) NEBNext Library Quant Kit for Illumina (NEB, Cat. No. E7630S) AmpureXP Purification Beads (Beckman Coulter, Cat. No. A63880) Illumina MiSeq Reagent Kit v2 (Illumina, Cat. No. MS-103-1001) PhiX Control V3 (Illumina, Cat. No. FC-110-3001) TapeStation System (Agilent, Model No. 4200) PCR plate (BIO-RAD, Cat. No. MLL9601) Adhesive PCR plate seal (BIO-RAD, Cat. No. MSB1001) Real-Time PCR (BIO-RAD, Model No. CFX Opus 96) MiSeq (Illumina, Model No. SY-410-1003)
Techniques: Sequencing, Generated, In Vitro, Clone Assay, Produced, Control, Reverse Transcription, Amplification
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