Streamlined and sensitive mono- and diribosome profiling in yeast and human cells

Ribosome profiling has unveiled diverse regulations and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected mRNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with Ordered Two-Template Relay, a single-tube protocol for sequencing library preparation that incorporates adapters by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved a myriad of distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling, and termination. Our optimized methods for mRNA footprint generation and capture provides a richer translatome profile using lower input and fewer technical challenges.