title: GSM9409583: PD1-Foxp3-CD4+T cells，anti-AXL Ab treatment, Day16 (biol rep3); Mus musculus; RNA-Seq
library_construction_protocol: Tumor tissues were harvested, minced into small fragments, and digested in RPMI 1640 containing 1 mg/mL collagenase type IV (Sigma-Aldrich) and 0.5 mg/mL DNase I (Roche) at 37 °C for 30 minutes. The resulting cell suspensions were passed through a 70 μm cell strainer to obtain single-cell suspensions. Cells were resuspended in FACS buffer (PBS supplemented with 2% FBS and 0.05% NaN₃), blocked with anti-mouse CD16/32 antibody (clone 2.4G2) for 20 minutes, and then stained with fluorophore-conjugated antibodies for 30 minutes at 4 °C. Target cell subsets for sequencing were subsequently sorted by flow cytometry, deposited into tubes containing 50 μL of lysis buffer, centrifuged, and immediately frozen at −80 °C. RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.