Figure EV4. mRNA concentration of transcripts of interest in G1-, S- and G2/M-phase measured with smFISH are shown in comparison to negative controls.

(A–F) Wild-type cells were incubated with or without smFISH probes against ACT1 (A–C) or MDN1 (D–F), ACT1, MDN1 (${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{YPD}}$ = 48, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{YPD}}$ = 54, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{YPD}}$ = 74, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCD}}$ = 26, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCD}}$ = 31, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCD}}$ = 30, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCGE}}$ = 26, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCGE}}$ = 23, and ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCGE}}$ = 49); no probes (${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{YPD}}$ = 49, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{YPD}}$ = 40, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{YPD}}$ = 43, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCD}}$ = 28, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCD}}$ = 27, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCD}}$ = 52, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCGE}}$ = 24, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCGE}}$ = 30, and ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCGE}}$ = 16). (G) Wild-type cells were treated with thiolutin for 80 min to inhibit global transcription prior to incubation with MDN1 probes. mRNA concentrations in G1, S, and G2/M were estimated by dividing the number of detected spots by the cell volume (${\mathrm{n}}_{\mathrm{G}1}^{MDN1}$ = 14, ${\mathrm{n}}_{\mathrm{S}}^{MDN1}$ = 42, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{MDN1}$ = 40, ${\mathrm{n}}_{\mathrm{G}1}^{MDN1\mathrm{,aftertranscr.inhibition}}$ = 57, ${\mathrm{n}}_{\mathrm{S}}^{MDN1,\mathrm{aftertranscr.inhibition}}$ = 40, and ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{MDN1,\mathrm{aftertranscr.inhibition}}$ = 34). (H–J) Wild-type cells expressing no mCitrine, as well as cells carrying an additional copy of the HTB1, HTB2, or ACT1 promoter driving mCitrine were incubated with smFISH probes against mCitrine. mRNA concentrations in G1, S, and G2/M were estimated by dividing the number of detected spots by the cell volume. Box plots represent median and 25th and 75th percentiles; whiskers indicate the 5th and 95th percentiles and symbols show outliers; HTB1prom-mCitrine (${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{YPD}}$ = 58, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{YPD}}$ = 49, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{YPD}}$ = 41, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCD}}$ = 54, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCD}}$ = 51, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCD}}$= 53, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCGE}}$ = 28, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCGE}}$ = 39, and ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCGE}}$ = 28); HTB2prom-mCitrine (${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{YPD}}$ = 37, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{YPD}}$ = 64, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{YPD}}$ = 59, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCD}}$ = 85, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCD}}$ = 59, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCD}}$ = 50, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCGE}}$ = 65, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCGE}}$ = 50, and ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCGE}}$ = 55); ACT1prom-mCitrine (${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{YPD}}$ = 35, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{YPD}}$ = 59, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{YPD}}$ = 41, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCD}}$ = 38, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCD}}$ = 75, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCD}}$ = 42, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCGE}}$ = 37, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCGE}}$ = 33, and ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCGE}}$ = 54); no mCitrine (${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{YPD}}$ = 15, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{YPD}}$ = 28, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{YPD}}$ = 37, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCD}}$ = 32, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCD}}$ = 30, ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCD}}$ = 48, ${\mathrm{n}}_{\mathrm{G}1}^{\mathrm{SCGE}}$ = 26, ${\mathrm{n}}_{\mathrm{S}}^{\mathrm{SCGE}}$ = 49, and ${\mathrm{n}}_{\mathrm{G}2\mathrm{M}}^{\mathrm{SCGE}}$ = 31).