GSM8416742: ENZA_early; Mus musculus; RNA-Seq
TRAMP-C1 allografts were resected from C57/BL6N-tumor bearing mice either 12 days (Early phase) or 24 days (Late phase) after the initiation of the treatment. Enzalutamide, Enzalutamide + anti-IL-23 and Enzalutamide + aCXCR2 therapies were started the day after surgical castration.For single-cell RNAseq, tissues were digested in Digestion Buffer composed by RPMI 1640 Normal medium, Collagenase D (1 mg/mL), DNAse (100 U/mL) and HEPES (25 mM), in the presence of RNase Inhibitor Murine (New England Biolabs). The cell suspension was incubated for 40 min at 37 °C on a rocker. The digestion was stopped adding Normal medium and cell suspension was filtered by a 100 mm cell strainer and kept on ice for 4 min. After further filtering, cell suspension was spun down at 1500 rpm for 5 min at 4°C and Cell subsets were enriched by FACS sorting. Flow Cytometry staining of tumors was performed using the following antibodies: anti-CD45-bv605 (clone 30-F11), and anti-CD11b-bv421 (clone M1/70); and anti-Gr1-APC (clone RB6-8C5), and 7AAD (eBioscience). The 7AAD negative cells were sorted as follows: i) CD45+/CD11b+/Gr1+; ii) CD45+/CD11b-/Gr1-; iii) CD45-. After counting, cells were mixed together in ratio 1:1:1 to perform single-cells sequencing. Single-cell suspensions were processed with Single Cell 3′ Reagent Kit v3 (Chromium Single Cell 3′ Library & Gel Bead Kit v3, Chromium Single Cell G Chip Kit, 10x Genomics) and Chromium Controller (10x Genomics) according to the protocol at the BIOS+ Genomics Facility in Bellinzona. The generated libraries were sequenced using Illumina NextSeq500 Systems accordingly with the 10x Genomics manual.