transcriptional profiling of cells sorted by rna abundance

nature methods | VOL.11 NO.5 | MAY 2014 | 549 The proposed RNA cell-sorting technique uses flow cytometry to measure the fluorescence of individual cells labeled with a single-molecule RNA FISH (smFISH) probe library13,14. As a proof-of-principle experiment, GFP transcripts were fluorescently labeled in cells that expressed the transgene under doxycycline control15 (Supplementary Fig. 1a–c). To assess the sorting potential of the labeled RNA signal, we measured singlecell RNA fluorescence distributions by flow cytometry, which revealed a clear separation of highand low-induction profiles (Fig. 1a and Supplementary Fig. 1b,c). Furthermore, we found the measured mRNA fluorescence to scale linearly with mRNA and protein abundance across a broad range of induction levels (Fig. 1b). We further confirmed the linearity of the labeled RNA fluorescence signal for a panel of endogenous genes by comparing the mean flow cytometry signal intensity with the average number of RNA molecules in iPSCs quantified by single-cell transcript counting14 (Fig. 1c). We note that the absolute number of gene transcripts expressed in stem cells is dependent on both genetic background and medium conditions (Supplementary Fig. 2). We then asked whether the observed RNA fluorescence signal provides an accurate measurement of single-cell transcript levels. For this purpose, we measured both GFP protein and labeled mRNA fluorescence in single cells and found a strong correlation (Pearson’s correlation coefficient (ρ) = 0.77; Supplementary Fig. 1d), which is consistent with the direct dependence of protein production on mRNA abundance. Additionally, the correlation between mRNA and protein was confirmed across a broad range of GFP induction levels (Supplementary Fig. 1e). Next we tested the single-cell precision of the proposed RNA measurement by differentially labeling the 3′ and 5′ ends of a single transcriptional target, Oct4-IRES-GFP fusion mRNA (where Oct4 is the gene Pou5f1 and IRES is an internal ribosome entry site), and found that the labels were strongly correlated at the single-cell level (ρ = 0.90; Supplementary Fig. 3a). Furthermore, we separately labeled Oct4 in mouse embryonic stem cells (mESCs) with either Alexa 594 or Cy5 fluorophores. We then mixed the differentially labeled cells and confirmed by flow cytometry that the two subpopulations were strongly anticorrelated (ρ = −0.81; Supplementary Fig. 3b). Having established measurements of both positive and negative correlation, we hypothesized that doxycycline-induced GFP expression would in principle be uncorrelated with every endogenous mRNA species in the genome. We confirmed this prediction across all measured GFP induction levels for the gene Oct4 (ρ = 0.1; Supplementary Fig. 3c). Finally, we sorted cells above and below each quartile of the Sox2 RNA fluorescence transcriptional profiling of cells sorted by rna abundance