Fig. 1

Ribo-seq workflow and representative gel images. A Ribo-seq overview: a Ribo-seq experiment consists of ribosome footprinting and sequencing library construction. B Workflow for custom Ribo-seq library construction. After size selection, ribosome footprints undergo end repair, ligation to a linker, removal of excess linkers, rRNA depletion, reverse transcription, cDNA purification, cDNA circularization, library PCR, and library purification. Among these steps, three (size selection of ribosome footprints, cDNA purification, and library purification) involve gel purifications and are highlighted by red boxes. For 20–30-nt ribosome footprints, the expected cDNA length would be 94–104 nt, and the expected library size would be 162–172 bp. C Size selection of ribosome footprints using a 15% TBE-urea gel. Lane 1: 30- and 28-nt marker (from the discontinued illumina Ribo-seq kit) and 25-, 21-, 17-nt marker (NEB microRNA marker). Lane 2: DynaMarker Prestain Marker for Small RNA Plus (Diagnocine). Lane 3: ribosome footprint sample. In this study, gel slices corresponding to the 20–30-nt range (marked by the bracket) were excised. D cDNA purification using a 10% TBE-urea gel. Lane 1: ssDNA ladder. Lane 2: cDNA sample. The bracket marks the expected cDNA length (94–104 nt), and the 74-nt band corresponds to the unused RT primer. E The resulting library after amplification with 11 cycles of PCR and resolved on an 8% TBE gel. Lane 1: 20-bp ladder. Lane 2: the library product; the bracket marks the expected library size (162–172 bp). The asterisk marks the product from an unextended RT primer, which should be avoided. F Fragment Analyzer profile showing enrichment of the expected library size. LM and UM are the vendor’s internal size markers