S1 nuclease mapping identifies the precise 5′ end of an mRNA transcript. A 5′-labelled ssDNA probe that overlaps the suspected transcript start is hybridised to extracted mRNA. S1 nuclease degrades single-stranded overhangs, leaving only the protected RNA:DNA hybrid. The length of the protected fragment, determined by denaturing PAGE alongside a sequencing ladder, pinpoints the transcription initiation site.