IMMAGINA Biotechnology S.r.l recently developed RiboLace (9 sample prep), a novel method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach.
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. Please note that RiboLace will be shipped in two separated boxes: one at +4°C and the second in dry-ice. The shipping is usually scheduled within 20 days from your order. Since RiboLace is a new product, we guarantee constant customer support. For questions: firstname.lastname@example.org.
Clamer et al., Active ribosome profiling with RiboLace. Cell Reports 2018, 25, 1097-1108.
Lauria et al. SMN-primed ribosomes modulate the translation of transcripts related to Spinal Muscular Atrophy bioRxiv, September 2019.
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.
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.
Scholtz et al. uORF-Tools—Workflow for the determination of translation-regulatory upstream open reading frames PLoSONE 2019, 14, e0222459.
Wang et al. Recent advances in ribosome profiling for deciphering translational regulation Methods 2020, 176, 46-54.
Sample preparation is crucial for a good periodicity of the P-site along the coding sequence.
i) CHX treatment of the cells is not mandatory and depends on your experimental setup. CHX is present in the RiboLace lysis buffer
ii) Flash-frozen samples/tissues give usually better results.
iii) 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.
iiii) 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.