GSM8862894: CN3; Homo sapiens; RNA-Seq
Buffer preparation: ST buffer was prepared as a 2x stock solution containing 292 mM NaCl, 20 mM Tris-HCL (pH 7.5), 2 mM CaCl2, and 42 mM MgCl2 in nuclease-free water. This served as a base for preparing ST wash buffer and TST lysis buffer. Lysis buffer (TST) was prepared by diluting the 2x ST buffer stock solution with nuclease-free water and adding the following reagents: Tween-20 (0.03% final), BSA (0.01% final) and recombinant RNAse inhibitor (final concentration: 0.25 U/µL). Nuclei isolation: Omental metastases samples were collected from seven HGSOC patients, snap frozen, and stored at -80°C. 60 to 100 mg of frozen tissue was used for each dissociation. Tissue samples were chopped in cold TST buffer using a razor blade until a homogenous pulp was obtained, followed by filtration through a 40 µm filter. The filtered nuclei solution was centrifuged at 500 g for 5 min at 4°C. The pellet was resuspended in cold ST buffer and filtered through a 20 µm sieve placed on a DNA low-bind microcentrifuge tube to remove smaller debris. The filter was rinsed with PBS-0.04% BSA and centrifuged. The nuclei were then resuspended in PBS-0.04% BSA and counted. Nuclei concentration was adjusted to 1,000 nuclei/µL by diluting with PBS-0.04% BSA. If the sample was clumpy, it was again strained through a 20 µm filter. The isolated nuclei were loaded onto a Chromium Next GEM Chip (10X Genomics) following the manufacturer's protocol at a concentration of 10,000 cells/nuclei per well. We used the Chromium Next GEM Single Cell 5' Reagent Kits, including Single Cell V(D)J Reagent Kits v1.1 with Chip G.