Clinical samples are vital for understanding diseases, but their scarcity requires refined research methods. Emerging single-cell technologies offer detailed views of tissue heterogeneity but need sufficient fully characterized tissues. We developed an optimized single-nuclei RNA sequencing (snRNA-seq) protocol to extract nuclei from just 15 mg of cryopreserved human tissue. Applied to four cancer tissues (brain, bladder, lung, prostate), it profiled 1550-7468 nuclei per tissue, revealing heterogeneity comparable to public single-cell atlases. This method enhances the use and sharing of rare, cryopreserved biospecimens, supporting research where sample quantity is limited and full tissue characterization is needed.