title: GSM8152621: NBM08; Homo sapiens; RNA-Seq
library_construction_protocol: We used the Tween with salts and TRIS (TST) tissue lysis buffer combined with the dounce and iodixanol density gradient medium. For nucleus extraction, Snap-frozen tumor tissue pieces were placed in a glass dounce homogenizer containing TST lysis buffer. After careful mechanical dissociation with the dounce pestle, followed by 5 minutes of incubation on ice in a total of 5 mL lysis buffer, the reaction was quenched by adding 5 mL wash buffer. The wash buffer consisted of 15 mL 10% bovine serum albumin (BSA, Sigma-Aldrich, cat. no. 05482-25G) dissolved in sterile nuclease-free water (Invitrogen, cat. no. 10977049); 70 μL of 100% glycerol (Sigma-Aldrich, cat. no. G5516), 30 μL Protector RNase inhibitor at 40 units/μL (Millipore Sigma, cat. no. 3335402001), and 15 mL sterile PBS (Gibco). The lysed suspension was passed through a 30 μm filter and centrifuged at 500g, 4°C, for 5 minutes, after which the pellet was resuspended in wash buffer, filtered, and centrifuged again. Next, the pellet was resuspended in 1 mL wash buffer, mixed with 1 mL 50% Optiprep solution (Iodixanol density gradient medium, Millipore Sigma, cat. no. D1556- 250). Optiprep was diluted in a buffer containing 20 mM tricine-KOH buffer at pH 7.8, 150 mM KCl, 5 mM MgCl2, and nuclease-free water. This 25% Optiprep/nucleus mix was layered on top of 2 mL 29% Optiprep and centrifuged at 10000g for 30 minutes at 4°C. The pellet was resuspended in a final nucleus suspension with 300 μL wash buffer and filtered through a 40 μm pipette tip filter. 10X Genomics Single cell 3' gene expression V2