RiboLace kit, improving ribosome profiling experiments

Immagina Biotechnology provides an innovative solution for ribosome profiling with RiboLace. Classical

ribosome profiling approches do not distinguish between fragments protected either by actively translating or

by inactive ribosomes.

Immagina's proprietary RiboLace technology allows 1-day selective extraction of ribosomes in active

translation and purification of ribosome protected fragments(RPFs). Suitable starting material can be lysates

of flash-frozen tissues or immortalized/primary cell cultures (>10,000 cells).

RiboLace kit is composed of two modules each of which can be independently purchased. RL001_mod 1 for

RPF extraction, and RL001_mod 2 for Illumina library preparation.

Features:

• Low RNA input requirements

• Strong enrichment of translated transcripts, which are functionally relevant for biological pathways of

  interest

• Capturing the active ribosomes is improving greatly the data to noise ration and increasing the

  concordance of transcriptomics data with the actual proteome

• Workflow improvements significantly reducing lab time and allowing for higher sample throughput

• Based on beads separation: possibility to run multiple samples in parallel for automated high-throughput

  experiments.

• No ultracentrifugation step

 Data Sheet (PDF)

RiboLace_module 1

Principle and procedure module 1

 Protocol (PDF)

RiboLace_module 2

Principle and procedure module 2

 Protocol (PDF) 

Tips;

1. Sample preparation is crucial for a good periodicity of the P-site along the coding sequence.

2. CHX treatment of the cells is not mandatory and depends on your experimental setup. CHX is present in

   the RiboLace lysis buffer

3. Flash-frozen samples/tissues give usually better results.

4. RiboLace lysis buffer does not contain protease inhibitors. If your sample is strongly sensitive to

   proteases, you can add EDTA free protease inhibitors to the lysis buffer.

5. During the preparation of the libraries, gel extraction steps are critical to reduce tRNAs and rRNAs

  contaminations. If the selection of the bands is not accurate, the deep of the sequencing needs to be

   improved to get enough reads from ribosome protected fragments.

References;

1. Clamer et al., Active ribosome profiling with RiboLace. Cell Reports 2018, 25, 1097-1108.

2. Lauria et al. SMN-primed ribosomes modulate the translation of transcripts related to Spinal Muscular Atrophy bioRxiv, September 2019.

3. Königs et al. SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly Nature Structural & Molecular Biology 2020, 27, 60–273.

4. Luoni et al. Whole brain delivery of an instability-prone Mecp2 transgene improves behavioral and molecular pathological defects in mouse models of Rett syndrome eLife 2020, 9, e52629.

5. Scholtz et al. uORF-Tools—Workflow for the determination of translation-regulatory upstream open reading frames PLoSONE 2019, 14, e0222459.

6. Wang et al. Recent advances in ribosome profiling for deciphering translational regulation Methods 2020,  176,  46-54.