Cell suspension reagent

The enriched CTC blood sample was centrifuged at 300 g for 10 min and concentrated to 70 $\mathsf{\mu }$ L. The cells were stained with the addition of the following markers and antibodies for 1 hour: CellTracker Orange (CTO) (Thermo Fisher, C34551), Calcein AM (Thermo Fisher, L3224), CD45 antibody- conjugated with Alexa 647 (Bio Legend, 304020), and CD31- conjugated with Alexa 647 (Bio Legend, 303111). 15 $\mathsf{\mu }$ L of RPMI with 10% FBS (Gibco) and 3 $\mathsf{\mu }$ L of RNase inhibitor (Thermo Fisher, N8080119) were also added to improve the viability and RNA quality of the cells. After incubation, 13 mL of PBS was added to dilute the staining reagents. The sample was spun down at 300 g for 10 min and concentrated to 45 $\mathsf{\mu }$ L. In order to achieve optimal buoyancy in an integrated fluidic circuit (IFC), 45 $\mathsf{\mu }$ L of CTCs was mixed with a 30 $\mathsf{\mu }$ L Cell suspension Reagent (Fluidigm, 101-0434) to achieve 75 $\mathsf{\mu }$ L of cell mix.