GSM9430052: HGSOC tissue, circASCOR high 3; Homo sapiens; RNA-Seq
Fresh ovarian cancer tissue samples (approximately 50–100 mg) were immediately dissected after surgical resection, rinsed 3 times with pre-chilled phosphate-buffered saline (PBS) containing 1% penicillin-streptomycin to remove residual blood and necrotic tissue, and cut into 1–2 mm³ pieces using sterile scalpels on ice. Tissue pieces were transferred into a 15 mL centrifuge tube containing 5 mL pre-chilled nucleus isolation buffer (NIB; 0.25 M sucrose, 25 mM KCl, 5 mM MgCl₂, 20 mM Tris-HCl pH 7.4, 1 mM DTT, 0.1% Triton X-100, 1× protease inhibitor cocktail). The mixture was gently pipetted 20–30 times with a 1 mL pipette tip to facilitate mechanical dissociation, followed by incubation on ice for 10 min. The homogenate was filtered sequentially through 70 μm and 40 μm cell strainers to remove undissociated tissue debris. The filtrate was centrifuged at 300 × g for 10 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 5 mL pre-chilled NIB, followed by a second centrifugation at 300 × g for 10 min at 4 °C. The pellet was resuspended in 1 mL pre-chilled NIB, and the nuclear suspension was layered onto a 30% sucrose cushion (prepared in NIB). After centrifugation at 1,500 × g for 20 min at 4 °C, the supernatant was discarded, and the purified nuclear pellet was resuspended in 500 μL pre-chilled PBS containing 0.1% BSA. Nuclear integrity was assessed using a hemocytometer under a phase-contrast microscope; only samples with >90% intact nuclei (round morphology, clear nuclear membrane, no obvious cytoplasmic contamination) were retained. The concentration of nuclei was adjusted to 1×10⁶ nuclei/mL for subsequent library construction. Library construction was performed using the 10× Genomics Chromium Single Cell 3′ Reagent Kit v3.1 following the manufacturer's standard protocols with minor modifications adapted for ovarian cancer nuclear samples:Single Nucleus Partitioning: Purified ovarian cancer nuclei (1×10⁴ nuclei per sample) were mixed with reverse transcription (RT) reagents and loaded onto the Chromium Chip G. The chip was processed on the Chromium Controller to generate Gel Bead-In-Emulsions (GEMs), where each GEM contained a single nucleus, a barcoded gel bead, and RT reaction mix.Reverse Transcription and cDNA Amplification:GEMs were incubated in a thermal cycler for RT reaction (53 °C for 45 min, 85 °C for 5 min, hold at 4 °C) to synthesize barcoded cDNA. Emulsions were then broken, and cDNA was recovered and purified using AMPure XP beads. Purified cDNA was amplified via PCR (98 °C for 3 min; 12–15 cycles of 98 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s; 72 °C for 5 min; hold at 4 °C) to obtain sufficient cDNA yield.Library Fragmentation, End Repair and Adapter Ligation:Amplified cDNA was fragmented to 200–300 bp using a Covaris M220 Focused-ultrasonicator. Fragmented cDNA was subjected to end repair, A-tailing, and ligation with indexed adapters (Illumina) according to the kit instructions. Adapter-ligated DNA was purified using AMPure XP beads to remove excess adapters and small fragments.Final Library Amplification and QC:The ligated products were amplified by PCR (98 °C for 3 min; 6–8 cycles of 98 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s; 72 °C for 5 min; hold at 4 °C). The final libraries were quantified using a Qubit 4 Fluorometer (Thermo Fisher Scientific) and their size distribution was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Only libraries with average fragment size of 250–350 bp and concentration >10 nM were used for sequencing.