Allen R. Buskirk Author Answered Nov 5, 2024
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland
It's important to keep the cells frozen during the harvesting and lysis so that translation doesn't continue until lysis is complete (when translation is stopped by antibiotics or high concentrations of magnesium). You could filter a culture, collect the pellet, and freeze it on liquid nitrogen, and then use glass bead lysis alternating vortexing and incubation in liquid nitrogen. Filtering causes some artifacts, as we show in our eLife paper, but it does give you the right number of ribosomes on each transcript, just skewed codon occupancy. I certainly do not recommend harvesting cultures by centrifugation.
I have not used kits for the library preparation stage, but there are companies that will make and sequence ribo-seq libraries for you, and there is a new shorter protocol for the library preparation by Nick Ingolia's lab: https://www.nature.com/articles/s41592-023-02028-1
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