title: GSM9510938: CD45+ cells, PBS  treatment, Day15; 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. Single-cell RNA-seq libraries were prepared using the Chromium Single Cell Reagent Kits v3 (10x Genomics), in accordance with the manufacturer's instructions. Cells were captured in droplets to target a specific cell recovery rate. Following the reverse transcription step, emulsions were broken and barcoded cDNA was purified with Dynabeads, followed by PCR amplification. The amplified cDNA was then used for constructing the gene expression library. For this construction, 50 ng of amplified cDNA was fragmented and end-repaired, double-size selected with SPRIselect beads, and sequenced on a NovaSeq platform (Illumina) to generate 150 bp paired-end reads, as per the manufacturer's instructions.
identifiers.namespace: GEO
identifiers.id: GSM9510938_r1