C1 cell suspension reagent

For MCF10CA single-cell RNA sequencing experiments, spheroids were dissociated as described above and resuspended in DMEM/F12 medium. Capture, full-length cDNA synthesis and amplification was performed on the C1 Single-Cell Auto Prep system for mRNA Seq (Fluidigm) using the IFC for up to 96 cells (medium size 10–17 µm). Cells at a concentration of 350 cells/µl were mixed with C1 Cell Suspension Reagent (Fluidigm) at a ratio of 4:1 immediately before loading on the IFC. Single-cell capture was assessed with an inverted brightfield microscope. Workflow and reagents for single-cell RNA extraction, reverse transcription (RT) and mRNA amplification (18 cycles) were used as described in the SMARTer Ultra Low RNA Kit (for Fluidigm C1). Sequencing libraries were generated with the Nextera XT kit (Illumina) according to an adapted Fluidigm protocol. Concentration and quality of cDNA and sequencing libraries was assessed by a fluorometer (Qubit) and by electrophoresis (Agilent Bioanalyzer high sensitivity DNA chips). Libraries of up to 24 cells were pooled and sequenced as 1 × 50-bp reads on an Illumina HiSeq 2000 machine.

To discriminate OHT and vehicle-treated Ezh2-conditional iMEFs, DiI (vehicle-treated) or DiO (OHT-treated) was added in growth medium for 4 h. Cells were then trypsinized and counted on a Vi cells counter (Beckman-Coulter). The average diameter of iMEFs was 13 μm. After mixing OHT- and vehicle-treated cells in equal proportions, 250,000 cells per milliliter were mixed at a 3:2 ratio in C1 cell suspension reagent (Fluidigm) before being loaded on a primed C1 Single-Cell Auto Prep Integrated Fluidic Circuit (Fluidigm). Cells were then visualized under an inverted fluorescent microscope (Leica) to assess viability and assignment of red (OHT-treated) and green (vehicle-treated) cells. Lysis, RT, and preamplifications were performed according to the manufacturer's protocol using Ambion Single Cell-to-CT kit (LifeTechnologies). Preamplified cDNA was analyzed by high-throughput qPCR on a Biomark-HD system (Fluidigm). The complete list of primers is in Supplemental Table S4. A qPCR primer pair designed on the region of the set domain that is deleted upon OHT treatment served as an independent genotype assignment and was found to closely match the color assignment. Only single cells with matching genotype/color assignments were considered for analysis.