Orthotopic Tumor Implantation and RNA Sequencing of Tumor-Associated CD11b⁺ Cells

The skull hole was sealed with bone wax, and the scalp was sutured. Mice were monitored daily and euthanized at planned endpoints or humane endpoints, including more than 20% body weight loss or severe neurological dysfunction.</p><p dir="ltr">At endpoint, tumor-bearing mouse brains were collected in cold RPMI 1640 medium, minced into approximately 1 mm3 pieces and dissociated using a gentleMACS brain tissue dissociation program.

Suspensions were filtered through a 70 um cell strainer, treated with red blood cell lysis buffer and stained for flow cytometry. Fc receptors were blocked with anti-CD16/CD32 antibody for 10 min at 4 °C. Cells were then incubated with fluorescent anti-CD11b antibody for 30 min at 4 °C in the dark and stained with DAPI to exclude dead cells. Debris was excluded by FSC/SSC gating, live DAPI-negative cells were selected, and CD11b+ cells were sorted by fluorescence-activated cell sorting.</p><p dir="ltr">Total RNA was extracted from sorted CD11b+ cells.

Libraries were prepared using the VAHTS Universal V6 RNA-seq Library Prep Kit and assessed on an Agilent 2200 Bioanalyzer. Sequencing was performed on a DNBSEQ-T7 platform with 150 bp paired-end reads. Clean reads were generated with fastp and quantified against the mouse GRCm38 reference using Salmon.

Counts and TPM values were used for principal component analysis and differential expression analysis with edgeR. </p><p><br></p>