RNA-seq of MDA-MB-231 with BCSC state sorting (A-BCSCs vs Q-BCSCs via SDorm system) and ENO1 silencing (siENO1 vs siNC)

Quiescent breast cancer stem cells (Q-BCSCs) can switch to an activated state (A-BCSCs). We established a dual-reporter system for stemness and quiescence (NANOG-EGFP + H2B-mCherry pulse-chase) to isolate A-BCSCs and Q-BCSCs from MDA-MB-231 cells. Strand-specific poly(A) RNA-seq was performed to compare A-BCSCs vs Q-BCSCs (n=3 per group). In a second dataset, ENO1 silencing (siENO1) was compared with a non-targeting control (siNC) in unsorted MDA-MB-231 cells (n=2 per group). Processed data are provided as separate experiment-specific gene-level matrices rather than a single unified matrix because different Ensembl gene annotation releases were used for quantification in the two experiments. For the A-BCSCs vs Q-BCSCs dataset, counts and FPKM matrices together with a matching gene annotation table are provided. For the siENO1 vs siNC dataset, counts and FPKM matrices together with a matching gene annotation table are provided. Previous reports have identified NANOG as a key stemness factor in tumor cells. Based on this, we established a stable fluorescent reporter system in MDA-MB-231 cells via lentiviral transduction, in which EGFP is driven by the NANOG promoter and expressed upon NANOG activation. To validate whether this system faithfully reflects cellular stemness, NANOG-EGFP positive and negative cells were sorted by FACS and subsequently analyzed by RNA-seq. Our results showed that the NANOG-EGFP positive population was enriched for stemness-related gene pathways, confirming that this reporter system can effectively indicate tumor cell stemness