Illumina HiSeq 2500 sequencing; Drug-resistant breast cancer stem cells enriched through one-dose chemo-therapy convey drug tolerance.
1x10^6 murine H8N8 mammary carcinoma cells were injected into the right abdominal mammary gland of female nulliparous WAP-T-NP8 mice. Once growing tumors reached a size of approx. 0.5 cm³, the animals were treated with a single intraperitoneal dose CAF chemotherapy (100 mg/kg cyclophosphamide, 5 mg/kg doxorubicin, and 100 mg/kg BW 5FU). Tumor cells were dissociated tumor tissues using the Tumor Dissociation Kit (Miltenyi Biotec) in combination with the gentleMACS Octo Dissociator with heaters (Miltenyi Biotec) according to the manufacturer's instructions. Finally, the obtained tumor cell suspensions were purified by magnetic-activated cell sorting (MACS) using CD45 (TIL) MicroBeadsin in combination with the Tumor Cell Isolation Kit (Miltenyi Biotec).  Total RNA was extracted from H8N8 cells (extracted from tumors growing in syngeneic animals) with Qiazol (Qiagen) following the manufacturer's recommendations.  mRNA sequencing libraries were generated with the NEXTflex™ Rapid Illumina Directional Kit according to the manufacturer's instructions. The size of the generated libraries was estimated on High Sensitivity DNA chip (Agilent) using a Bioanalyser 2100 (Agilent).