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US 12,467,048 B2Granted
Methods for mapping personalized translatome
AssigneeNorthwestern University
InventorsQianru Li, Haiwang Yang, Zhe Ji
Life of this patent
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Abstract
A low-input RNase footprinting approach is described for the rapid quantification of ribosome-protected fragments with 1,000-100,000 cells. The assay uses a simplified procedure for capturing ribosome-RNA complexes based on optimized RNase digestion. It simultaneously maps cytosolic and mitochondrial translation with single-nucleotide resolution.
Claims22
A method of RNAse footprinting comprising:
- (a) contacting a biological sample comprising cells with an RNAse in an amount sufficient to selectively protect first RNA fragments bound to a translational ribosome in a coding region, while digesting each of second RNA fragments bound by a RNA binding protein other than the translation ribosome in the coding region, (b) sequencing the first RNA fragments, and (c) mapping a ribosome profile for each of the first RNA fragments, wherein the number of cells in the biological sample is less than about 100,000, and wherein said method does not comprise ribosome isolation by centrifugation or immunoprecipitation.
A method of preparing open reading frame-coding RNA fragments from a biological sample comprising:
- (a) contacting the biological sample with an RNAse in an amount sufficient to selectively protect first RNA fragments bound to a translational ribosome in a coding region from digestion by the RNAse, while digesting each of second RNA fragments bound by a RNA binding protein other than the translation ribosome in the coding region, (b) isolating the first RNA fragments without centrifugation, and (c) sequencing the first RNA fragments coding an open reading frame.
A method of low input RNase footprinting comprising:
- (a) contacting a biological sample comprising 50,000 or less cells with an RNase in an amount sufficient to selectively protect first RNA fragments bound to a translational ribosome in a coding region, while digesting each of second RNA fragments bound by a RNA binding protein other than the translation ribosome in the coding region, (b) sequencing the first RNA fragments, and (c) mapping a ribosome profile for each of the first RNA fragments, wherein said method does not comprise centrifugation.
Dependencies are inferred from the claim text.