Abstract
We have developed protocols for rapidly quantifying the band intensities from nucleic acid chemical mapping gels at single-nucleotide resolution. These protocols are implemented in the software SAFA (semi-automated footprinting analysis) that can be downloaded without charge from http://safa.stanford.edu. The protocols implemented in SAFA have five steps: (i) lane identification, (ii) gel rectification, (iii) band assignment, (iv) model fitting and (v) band-intensity normalization. SAFA enables the rapid quantitation of gel images containing thousands of discrete bands, thereby eliminating a bottleneck to the analysis of chemical mapping experiments. An experienced user of the software can quantify a gel image in ∼20 min. Although SAFA was developed to analyze hydroxyl radical (·OH) footprints, it effectively quantifies the gel images obtained with other types of chemical mapping probes. We also present a series of tutorial movies that illustrate the best practices and different steps in the SAFA analysis as a supplement to this protocol.
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Acknowledgements
This work was funded by National Institutes of Health Grants P01-GM66275 to D.H., U54-GM072970 (National Centers for Biomedical Computation) to R.B.A., P41-EB0001979 to M.B., and K99/R00 (GM079953) award to A.L. and the NSF 0443508 for the RNA Ontology Consortium. Q.V. acknowledges the Howard Hughes Medical Institute for support.
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Laederach, A., Das, R., Vicens, Q. et al. Semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments. Nat Protoc 3, 1395–1401 (2008). https://doi.org/10.1038/nprot.2008.134
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DOI: https://doi.org/10.1038/nprot.2008.134
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