GSM8552409: NL106; Homo sapiens; RNA-Seq
The samples were chopped on ice into 1-2 mm³ pieces, transferred to cryovials, snap-frozen in liquid nitrogen for 5 minutes, and then stored in an ultra-low temperature freezer and liquid nitrogen.. Then transfer the tissue from the cryovial to a Dounce homogenizer and add 500 μl of pre-chilled lysis buffer to thaw. Use a small and a tight pestle to press the tissue 15-20 times and 5-10 times, respectively, to create a homogenate. Add 700 μl of nuclear wash buffer, mix, and filter through a 70 μm cell strainer. Add 1 ml of 50% iodixanol to the filtrate, mix gently, and layer onto a gradient solution of 30% and 33% iodixanol. Centrifuge at 10,000×g for 20 minutes at 4°C. Extract the white nuclear layer at the interface, add nuclear wash buffer, mix, and centrifuge at 500×g for 8 minutes at 4°C. Transfer the supernatant, resuspend the nuclei, and repeat the wash. Filter through a 40 μm filter, centrifuge, resuspend the nuclei, and adjust the concentration to 700-1200 nuclei/μl using a microscope. Cellular suspensions were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs). Libraries were generated and equenced from the cDNAs with Chromium Next GEM Single Cell 3' Reagent Kits v3.1.