Mass cytometry analysis

EQ^TM Four Element Calibration Beads (Fluidigm) were added to the cell suspension at a 1:10 ratio (v/v). Samples were analyzed on a Helios mass cytometer (Fluidigm). The manufacturer’s standard operation procedures were used for acquisition at a rate of ~200 cells per second. After data acquisition, all .fcs files from the same barcoded sample were concatenated. Data were then normalized, and bead events were removed⁴¹. Doublets were removed, and cells were de-barcoded into their corresponding wells using a doublet-filtering scheme and single-cell deconvolution algorithm⁴². Subsequently, data were processed using Cytobank (http://www.cytobank.org/). Additional gating on the DNA channels (¹⁹¹Ir and ¹⁹³Ir) was used to remove remained doublets, debris, and contaminating particulates. Manual gating was performed on IdU, cyclin B1, p-HH3, and p-RB to identify cell-cycle stages³².