RNase H cleaves RNA in RNA-DNA duplexes. It is present in all domains of life as well as in multiple viruses and is essential for mammalian development and for HIV replication. Moreover, human RNase H1 is essential for the therapeutic effect of DNA-like antisense oligonucleotides. Here, I will our efforts to understand the sequence preferences of HIV-1, human and E. coli RNase H enzymes using sequencing-based methods. Our findings can potentially be used to improve the design of RNase H-recruiting antisense oligonucleotides and show that sequence preferences of HIV-1 RNase H have shaped evolution of the HIV-1 genome and may contribute to the conserved use of tRNA-Lys3 as primer during viral replication.