Single-cell multi-omics can provide a unique perspective on tumor cellular heterogeneity. Most previous single-cell-whole-genome-RNA-sequencing (scWGS-RNA-seq) methods require physical separation of DNA and RNA, which makes these methods labor-intensive and technically demanding, time-consuming, or requiring special devices. And they are not applicable to frozen samples that cannot generate intact single-cell suspensions. We have developed scONE-seq, a versatile method for simultaneous transcriptome and genome profiling of single cells or single nuclei. Compared with existing methods, scONE-seq is a one-tube reaction that eliminates loss due to DNA and RNA separation, and thus is highly scalable and compatible with frozen biobanked tissue. We benchmarked scONE-seq against existing methods using fresh and biobanked samples and demonstrated superior performance, applying it to a 2-year-frozen astrocytoma sample and identifying a unique transcriptionally normal-like tumor clone. scONE-seq makes it possible to perform single-cell multi-omics interrogation with ease on the vast quantities of biobanked tissue. We also have a Git-hub repository : https://github.com/0YuLei0/scONE-seq-data-processing