Introduction
Ribosome‐profiling (Ribo-seq) captures ribosome-protected ∼28–34-nt mRNA fragments, providing a nucleic-acid-level view of translation. Since the archetypal Ingolia protocol was introduced in yeast in 2009, multiple variants have appeared to answer distinct biological questions or solve technical bottlenecks. In a previous blog we focused our attention on the utilization of small RNA library preparation methods to sequence the fragments obtained. Here we will be reviewing the five most widely used protocols to prepare those ribosome-protected mRNA fragments, outlining key steps, major advantages and trade-offs of each.
Classical monosome Ribo-seq
The canonical Ingolia protocol begins by arresting elongating 80S ribosomes in vivo with cycloheximide. Cells or tissue are rapidly lysed under conditions that preserve ribosome–mRNA complexes, and the lysate is digested with a single-strand nuclease (typically RNase I or MNase). Everywhere except where the ribosome occludes the mRNA, the transcript is shredded; the remaining ~28–34 nt “footprints” remain bound within monosomes. These complexes are then separated on a 10–50% sucrose gradient, the monosomal peak is collected, and the protected fragments are purified by denaturing PAGE before cDNA library preparation.
Because every actively elongating ribosome deposits one protected fragment, the resulting reads furnish genome-wide, single-codon maps of ribosome occupancy. Triplet periodicity allows unambiguous P-site assignment, enabling observation and quantification of events such as translation efficiency, pausing at specific codons, and frame maintenance. The method is species-agnostic with only minor tweaks (e.g., substituting MNase for RNase I in bacteria), but practitioners must account for cycloheximide-induced artefacts and rRNA contamination during analysis.
Initiation-focused Ribo-seq (GTI-seq, QTI-seq, and TIS-seq)
To interrogate initiation rather than elongation, cycloheximide is replaced by a drug that arrests ribosomes at, or immediately after, start-codon recognition. Lactimidomycin binds elongating ribosomes but leaves scanning 80S initiation complexes untouched until they engage the first AUG, thereby producing a sharp pile-up at translation start sites (GTI-seq). Alternatively, a brief harringtonine pulse allows scanning complexes to run-off and stall just after first peptide-bond formation, giving the closely related QTI-seq and TIS-seq read-outs. Downstream steps (nuclease digestion, gradient isolation, PAGE purification, and library prep) mirror the classical workflow.
The distinctive, sub-codon-wide peaks generated by these inhibitors enable single-nucleotide mapping of canonical and non-AUG initiation sites, making it possible to catalogue upstream open reading frames (uORFs), downstream open reading frames (dORFs), and leader-truncated isoforms with high sensitivity. Because the inhibitor window is brief (seconds to minutes), quantitative interpretation must consider drug-induced stress responses and the need for precise timing. Nevertheless, when paired with a classical monosome dataset from the same sample, initiation-focused Ribo-seq provides a complete delineation of ORF boundaries.
Translation-complex profiling (TCP-seq and Sel-TCP-seq)
TCP-seq expands the snapshot to include scanning and pre-initiation complexes. Cells are flash crosslinked with formaldehyde, immobilising 43S pre-initiation complexes, 48S initiation complexes, and 80S monosomes on their respective mRNA segments. After lysis, a deep sucrose gradient separates 40S, 60S, and 80S containing fractions; each is individually digested with nuclease, and footprints are purified and sequenced. In the selective (Sel-TCP-seq) variant, immunoprecipitation of initiation factors such as eIF3 or eIF4G is performed prior to footprinting, enriching factor-bound sub-populations.
Footprints derived from 40S complexes map the paths of scanning ribosomes, while 80S footprints report elongation on the same transcript, allowing direct estimation of scanning velocities versus elongation rates under identical physiological conditions. This dual view makes TCP-seq invaluable for studying how uORFs gate dORF expression, how initiation factors redistribute during stress, or how leader length modulates scanning pausing. The cost is a technically demanding, multi-gradient workflow that requires high input material and careful optimisation of crosslinking to balance complex capture against RNA integrity.
Active-ribosome pulldown (RiboLace™)
RiboLace™ (Immagina) replaces gradient ultracentrifugation with a chemoselective enrichment step. Lysates are incubated with magnetic beads coated with “3P,” a puromycin analogue that occupies the ribosomal A-site only in actively elongating ribosomes. Once bound, the complexes are magnetically collected, extensively washed, and then digested with nuclease directly on the beads. Protected fragments are released, size-selected (often using SPRI rather than PAGE), and converted to sequencing libraries in a same-day workflow.
Because enrichment occurs before nuclease digestion, rRNA- and tRNA-derived fragments are markedly reduced, improving library complexity and the signal-to-noise ratio of coding footprints. The method scales to nanogram-level inputs, making it attractive for primary tissue biopsies, rare cell populations, or high-throughput screens. However, it selectively captures elongating ribosomes with an accessible A-site, under-representing stalled, collided, or initiation complexes; and reliance on proprietary reagents can raise costs and may limit protocol flexibility.
Collision and queue-aware profiling (Disome-seq / Disome-profiling)
Disome-seq exploits the fact that two ribosomes stacked nose-to-tail protect a ~58–62 nt fragment, roughly twice the length of a monosomal footprint. Cells are harvested without CHX (or with only a brief pulse), lysates are digested gently so that adjacent ribosomes are not separated, and higher-density sucrose gradients enrich the heavier disome peak. After footprint purification and library prep, read lengths distinguish disomes from monosomes during alignment.
Positions at which disome reads accumulate mark sites of ribosome traffic jams—poly-basic stretches, poly-proline motifs, stop-codon queues, or mRNA lesions that trigger No-Go Decay. Comparing disome density with monosome occupancy separates genuine molecular roadblocks from CHX-induced artefacts and enables kinetic modelling of queue formation. Because collision footprints form only a few percent of total reads, deep sequencing and meticulous nuclease optimisation are essential; yet the resulting data uniquely illuminate co-translational quality-control pathways that are invisible to conventional Ribo-seq.
Protocol comparison
| Protocol | Key benefits | Key drawbacks |
|---|---|---|
| Classical monosome Ribo-seq | Genome-wide, single-codon resolution; broadly transferable across species; extensive QC metrics and community benchmarks. | Cycloheximide can induce artifactual pausing; provides no information on initiation events; libraries often dominated by rRNA fragments; labor-intensive PAGE and gradient steps. |
| GTI-seq / QTI-seq / TIS-seq | Precisely maps canonical and non-AUG start codons; reveals uORFs/dORFs and alternative leaders; complements classical profiling for ORF annotation. | Requires tight drug-pulse timing and dose optimisation; inhibitors trigger stress responses that reshape initiation; very short footprints risk loss during size selection. |
| TCP-seq (and Sel-TCP-seq) | Captures scanning 40 S, 48 S, and elongating 80 S complexes simultaneously; links specific initiation-factor occupancy with individual mRNAs; enables joint modelling of scanning and elongation kinetics. | Multi-day, technically demanding workflow with formaldehyde crosslinking and multiple gradients; high input (≥10⁸ cells) typically required; larger size window increases rRNA carry-over and reduces library complexity. |
| RiboLace™ (Immagina) | Gradient-free, gel-free workflow completed in hours; works with nanogram-level or clinical samples; strong enrichment for actively elongating ribosomes and improved signal-to-noise. | Relies on proprietary biotin-puromycin reagents, increasing cost; under-represents stalled or collided complexes lacking an accessible A-site; initiation and collision biology require additional protocols. |
| Disome-seq/Disome profiling | Specifically detects stacked ribosomes, pinpointing sites of traffic jams, poly-basic or stop-proximal stalls, and No-Go Decay triggers; distinguishes genuine pauses from CHX artifacts. | Disome footprints constitute a small fraction of total reads, demanding deep sequencing; nuclease digestion must be finely tuned. Over-digestion destroys disomes, under-digestion obscures monosome signal; interpreting collision signal requires modelling both translation speed and ribosome density. |
Concluding remarks
Choosing the most appropriate Ribo-seq method should follow directly from the biological question that needs to be answered. Classical monosome profiling remains the workhorse for differential ribosome occupancy measurements, whereas GTI- or QTI-seq is indispensable when the aim is to catalogue start codons with single-nucleotide precision.
If the objective is to dissect scanning dynamics or factor-specific initiation events, TCP-seq excels by capturing 40 S pre-initiation complexes in addition to elongating 80 S ribosomes. For rapid, low input, or clinically precious samples, the bead-based RiboLace™ workflow (Immagina) offers a streamlined route that enriches for actively translating ribosomes, while Disome-seq uniquely illuminates sites of ribosome traffic jams and co-translational quality control.
In practice, many laboratories now combine protocols. For example, pairing GTI-seq with conventional monosome profiling to refine ORF boundaries, or using RiboLace enrichment before Disome-seq to boost collision signal. Whichever protocol is chosen, computational analysis must be matched accordingly: specialised aligners are required for disome footprints, and dedicated TIS callers for initiation-focused libraries, with modern platforms such as RiboSeq.org providing protocol-specific quality-control dashboards. Looking ahead, emerging variants such as proximity-labelled APEX-Ribo, single-cell droplet Ribo-seq, and AI-assisted footprint deconvolution, promise to expand the resolution and scope of translational profiling still further, making Ribo-seq an increasingly versatile toolbox for probing every stage of the mRNA translation cycle.
Regardless of the methodology used to isolate ribosome-protected mRNA fragments, their size is ∼28–34-nt, which is ideal for downstream sequencing using library preparation protocols developed for Small RNA-Seq.
References:
- Ingolia N.T., Ghaemmaghami S., Newman J.R.S., & Weissman J.S. (2009). Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science, 324(5924), 218-223. https://doi.org/10.1126/science.1168978 (Science)
- Lee S., Liu B., Lee S., Huang S.X., Shen B., & Qian S-B. (2012). Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution. Proceedings of the National Academy of Sciences USA, 109(37), E2424-E2432. https://doi.org/10.1073/pnas.1207846109 (PubMed)
- Gao X., Wan J., Liu B., Ma M., Shen B., & Qian S-B. (2015). Quantitative profiling of initiating ribosomes in vivo. Nature Methods, 12(2), 147-153. https://doi.org/10.1038/nmeth.3252 (Nature)
- Archer S.K., Shirokikh N.E., Beilharz T.H., & Preiss T. (2016). Dynamics of ribosome scanning and recycling revealed by translation complex profiling. Nature, 535(7611), 570-574. https://doi.org/10.1038/nature18657 (PubMed)
- Clamer M., Tebaldi T., Lauria F., Bernabò P., Gómez-Biagi R.F., et al. (2018). Active ribosome profiling with RiboLace. Cell Reports, 25(4), 1097-1108.e5. https://doi.org/10.1016/j.celrep.2018.09.084 (PubMed)
- Zhao T., Chen Y-M., Li Y., Wang J., Chen S., Gao N., & Qian W. (2021). Disome-seq reveals widespread ribosome collisions that promote cotranslational protein folding. Genome Biology, 22, 16. https://doi.org/10.1186/s13059-020-02256-0 (genomebiology.biomedcentral.com)
