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: email@example.com.
Reference: Clamer et al., Active Ribosome Profiling with RiboLace. Cell Rep., 2018, Oct 23;25(4):1097-1108.e5.
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.