Gene expression profile at single cell level of BT-549 TNBC Spheroid cultures with inducible expression of CTL or MUC1 targeting knockdown

The MUC1-C protein evolved in mammals for adaptation of barrier tissues to loss of homeostasis. Prolonged activation of MUC1-C in settings of chronic inflammation promotes lineage plasticity, epigenetic reprogramming and the cancer stem cell (CSC) state. The effects of MUC1-C on the metabolism of CSCs remain unexplored. We used single cell RNA sequencing (scRNA-seq) to analyze the diversity of BT-549 spheroid cultures with and without knockdown of MUC1 to examine the effects of MUC1 on CSC renewal and metbolic states. Pelleted single-cell suspensions of BT-549 spheroid cultures were washed in 1,000 ul of PBS plus 0.4% BSA, transferred to low-retention microcentrifuge tubes (Fisher Scientific, Hampton, NH, USA), and then centrifuged for 10 minutes at 300g at 4C. Pellets were resuspended in 800 ul of PBS plus 0.4% BSA, and cells were counted by eye using INCYTO C-Chip Neubauer Improved Disposable Hemacytometers (VWR International Ltd., Radnor, PA, USA). A total of > 10,000 cells per sample were loaded per channel of the Chromium Next GEM Chip K for processing on the 10x Chromium Controller (10x Genomics, Pleasanton, CA, USA) followed by cDNA generation and library construction, as per manufacturer’s instructions (Chromium Next GEM Single Cell 5ʹ Reagent Kits v2 User Guide, Rev E). Libraries were normalized and pooled for sequencing on an Illumina NextSeq 500 system using a 150 cycle Mid-Output flow cell (Illumina, Inc., San Diego, CA, USA) with run parameters 26, 10, 10, 90. Sequenced libraries were demultiplexed using cellranger mkfastq (Cell Ranger v6.1.1). As part of the cellranger count pipeline, demultiplexed libraries were aligned to the human transcriptome (excluding introns) based on the 10X Genomics pre-computed human reference, GRCh38-2020-A. Both raw and cell-associated gene expression feature matrices (cell barcodes x gene counts) were output for use in downstream analyses.