GSM9518584: peritoneal macrophage, ID8 chemotherapy model, high fat  diet, RNAseq [HFD2]; Mus musculus; RNA-Seq
Peritoneal lavage was performed by injecting 5 mL of ice-cold phosphate-buffered saline (PBS) into the peritoneal cavity of euthanized mice. The lavage fluid was collected and centrifuged at 300 × g for 5 minutes at 4°C. The resulting cell pellet was resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were then incubated at 37°C in a 5% CO₂ atmosphere for 2 hours to allow for adherence. Non-adherent cells were removed by gentle washing with warm PBS. The adherent cells (peritoneal macrophages) were harvested using a cell scraper for subsequent analysis. For flow cytometry verification, cells were stained with antibodies against CD45 and F4/80 to identify the macrophage population. Data acquisition was performed on a flow cytometer, and analysis was conducted using appropriate gating strategies to exclude debris and doublets. Total RNA was isolated from peritoneal macrophages harvested from the ID8 syngeneic ovarian cancer model at specified time points using the RNeasy Mini Kit (Qiagen). To ensure high‑quality RNA, cell suspensions were treated with a debris removal solution (Miltenyi Biotec, Cat# 130‑109‑398) prior to extraction for efficient clearance of cellular debris. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.