Uncover a direct role of RNA in DNA double-strand break (DSB) repair in human cells. Plasmid systems having different RNA sequences or different levels of transcription were used to do a transient genetic assay in human cells. DSBs were induced on the plasmid constructs by CRISPR-Cas9 system, and the result of DSB repair was detected by Next-Generation Sequencing (NGS). Our customized NGS library preparation method enables to deeply sequence the DSB locus.