GSM8890097: Double-Positive1; Homo sapiens; RNA-Seq
Nuclei were isolated as previously described (refer to "Method section" for a detailed protocol). Briefly, frozen tissue was homogenized in 500 µl of lysis buffer (Nuclei Pure prep; NUC201-1KT, Sigma) using a disposable Eppendorf tube and incubated on ice for 12 min. The lysate was filtered through a 70 µm filter to remove debris and centrifuged at 500 g for 5 min at 4°C to pellet nuclei. The supernatant was discarded, and nuclei were resuspended in nuclei wash buffer, then filtered through a 40 µm filter. After another centrifugation at 500 g for 5 min, the pelleted nuclei were resuspended in 250 µl of nuclei wash buffer mixed with 250 µl of 50% iodixanol. This mixture was layered onto 500 µl of 29% iodixanol and centrifuged at 5000 g for 20 min at 4°C. For cell counting, 2 µl of acridine orange was added to 18 µl of the sample, analyzed using a Luna Countess to count nuclei, and the suspension was adjusted to the desired concentration for further experiments. Libraries were prepared using the Chromium Single Cell 3′ Library and Gel Bead v3 kit according to manufacturer's instructions. cDNA libraries were sequenced using the Illumina NovaSeq 6000 system at a minimum sequencing depth of 30,000 paired-end reads per nucleus.