20 min. Finally, 200 μl of PBS was added to the cell suspension and the samples were centrifuged at 1,800 r.p.m. at 4 °C for five minutes. Cell suspensions were stored in 1% PFA in PBS at 4 °C until analysis. The following antibodies were used: CTIP2 (rat, 1:100; Abcam: AB18465), TBR1 (rabbit, 1:100; Abcam: AB31940), BRN2 (mouse, 1:50; Millipore: MABD51), SATB2 (rabbit, 1:50; Epitomics, 2819-1), CUX1 (mouse, 1:100; Abnova: H00001523-M01). The anti-CUX2 antibody was generated in rat, tested in human fetal cortex and used at dilution of 1:100. The following secondaries were used: anti-rat Alexa 647, anti-mouse PE, anti-rabbit Alexa 488 (dilution 1:5,000). The LSRII system (BD Biosciences) was used for data acquisition, while data analysis was performed with FlowJo (Treestar). Cellular events were gated for singlets (FSC-A versus FSC-H) to exclude potential cell clumps. Dead cells were then excluded from singlet population by elimination of cells that stained positive for Live/dead Blue. Negative threshold gates were defined with control samples (secondary antibodies alone). Example FACS plots are shown in Supplementary Figure 2.
