Abstract: Methods, compositions, and kits for simultaneously generating RNA and DNA sequencing libraries are disclosed. In particular, the disclosed methods allow high-throughput amplification and sequencing of both RNA and DNA from a single sample source without the need to divide the sample to separate nucleic acids from each other. All of the preparation steps from harvesting nucleic acids from cells or tissue to preparing RNA and DNA sequencing libraries can be performed with the RNA and DNA pooled together. This technology streamlines generation of DNA and RNA sequencing data in combination and makes possible comprehensive genomic and transcriptomic profiling of a cell population concurrently.