GSM9813927: lsy004_p; Homo sapiens; RNA-Seq
Fresh PDAC tissues were placed in sterile RNase‑free dishes containing ice‑cold calcium‑ and magnesium‑free 1× PBS. Tissues were minced into approximately 0.5 mm² pieces, washed with PBS, and carefully dissected to remove blood clots and adipose tissue. Dissociation was performed in enzyme solution at 37°C with shaking at 100 rpm for 20 min. Digestion was stopped by adding 1× PBS supplemented with 10% fetal bovine serum, followed by gentle pipetting 5–10 times. The suspension was passed through a 70 μm cell strainer and centrifuged at 300×g for 5 min at 4°C. The cell pellet was resuspended in 100 μL of 1× PBS containing 0.04% BSA, mixed with 1 mL of 1× red blood cell lysis buffer, and incubated at room temperature or on ice for 2–10 min to eliminate erythrocytes. After centrifugation (300×g, 5 min, room temperature), dead cells were removed using the Miltenyi Dead Cell Removal Kit according to the manufacturer's instructions. The resulting single‑cell suspension was washed twice with 1× PBS/0.04% BSA, resuspended in 50 μL of the same buffer, and assessed for viability by trypan blue exclusion. Only suspensions with >85% viability were used; cell counts were determined using a Countess II Automated Cell Counter, and concentrations were adjusted to 700–1200 cells/μL. Single‑cell suspensions were loaded onto the 10× Genomics Chromium platform to generate gel beads in emulsion using the Chromium Single‑Cell 3′Reagent Kit (v3) following the manufacturer's protocol. Subsequent cDNA amplification and library construction were performed according to standard procedures. Libraries were sequenced on an Illumina NovaSeq 6000 system with paired‑end 150 bp reads, targeting a minimum depth of 20,000 reads per cell. Sequencing was carried out by LC‑Bio Technology Co., Ltd. (Hangzhou, China).