For optimal nucleotide sequence mapping coverage, it is recommended to perform RNase 4 digests under denaturing conditions to limit the possible influence of RNA secondary structure. A digest reaction containing a final urea concentration at 1 M, incubated at 37 °C for 1 h, will ensure most RNA is available for endonucleolytic cleavage. RNase 4 endoribonuclease activity tolerates up to 3 M urea or 50% formamide (v/v). T4 Polynucleotide Kinase end repair activity tolerates up to 3 M urea or 25 % formamide (v/v).
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