Should I treat my SLAMseq RNA samples with DNase I and should this be done before or after alkylation with iodoacetamide?

DNase I treatment of your RNA is optional. If the sample must be DNase I treated, heat inactivation should be avoided, and the enzyme should be deactivated by other means such as (acidic) phenol / chloroform extraction, or silica column purification. We would best advise that DNase I treatment of RNA from SLAMseq kinetics experiments be performed after alkylation with iodoacetamide, when the modified nucleotides are more stable.