title: RNA-seq of mouse EO771 triple-negative breast cancer model with stemness inhibitors: salinomycin, SB-431542, JIB-04, napabucasin, and their quadruple combination against an untreated control
design: RNA-seq of mouse EO771 triple-negative breast cancer model with stemness inhibitors: salinomycin, SB-431542, JIB-04, napabucasin, and their quadruple combination against an untreated control
library_name: Sample 1_p
library_construction_protocol: The syngeneic EO771 mouse breast cancer model was used to evaluate the in vivo effects of stemness pathways inhibitors. For tumor implantation, female C57BL/6 mice (n=18, 8–12 weeks old) were anesthetized with 2% isoflurane (Vetpharma, cat. no. 90882) and injected subcutaneously in the right flank with 1 × 10^6 EO771 cells (>95% viability) suspended in 100 μL of PBS. After a 10-day period for tumor establishment, the mice received three doses of the specified drugs, either as monotherapy or in quadruple combination (n=3 per group), administered every three days. The following doses were used for the treatments: salinomycin (5 mg/kg), SB-431542 (10 mg/kg), JIB-04 (110 mg/kg), and napabucasin (20 mg/kg). All drugs were administered via intraperitoneal injection. Mouse were monitored for overall health and body weight. One day after the last dose, the mice were sacrificed in a CO₂ chamber, and the tumors were collected for bulk RNA-seq analysis. Tumor samples were immediately processed upon collection.  Up to 30 µg of tumor tissue was mechanically dissociated in TRIzol (Invitrogen, cat.no 15596026), and RNA was isolated using the RNeasy Mini Kit (Qiagen, cat. no. 74106) following the manufacturer's instructions. RNA quantity and quality were assessed using a NanoDrop 2000 spectrophotometer. Tumor RNA samples were processed using the Bulk RNA Barcoding and Sequencing (BRB-seq) platform with the Mercurius BRB-seq kit (Alithea Genomics, v.0.1.61, cat. no. 10813). Approximately 50 ng of total RNA was used per sample to generate BRB-seq libraries. Bulk RNA barcoding and sequencing (BRB-seq), and pre-processing of the data (demultiplexing and alignment) was performed by Alithea Genomics, as described previously Alpern, D. et al. BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing. Genome Biol. 20, 71 (2019).