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URL: https://dx.doi.org/DOI:10.17504/protocols.io.hdfb23n

Authors: Steven J. Burgess

Summary: Protocol for DNase I digestion of nuclei for 'double hit' DNase-SEQ analysis adapted from doi:10.1038/nmeth.2762As a general guide DNA should show moderate to light smearing after completing the protocol as seen the gel image in Figure 1.Figure 1: A representative 2% (w/v) LMP agarose gel of DNAse I treated nuclei (~2x108) from Sorghum bicolor. To each sample the following amounts of DNase I were added: none, 7.5U, 12.5U.Over-digested samples will result in a high background signal from unspecific cutting of DNA and should be avoided. An example of an over-digested sample is provided in lane 5 of Figure 2.Figure 2: A representative 2% (w/v) LMP agarose gel of DNAse I treated nuclei (~2x108) from Sorghum bicolor. To each sample the following amounts of DNase I were added: none, 5,U 7.5U,10U, 12.5U.Gel fragments from ~50-600bp should be excised and DNA purified. From experience taking only smaller fragments (50-200 bp) caused difficulties in library preparation.

Affiliations: University of Cambridge

Version: 1

Publication Date: 2017

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