Characterizing and inferring quantitative cell cycle phase in single-cell RNA-seq data analysis

Overview of study design. We collected two types of data from the same single cells using FUCCI-expressing iPSCs: in situ fluorescence images and scRNA-seq. After quality control, we obtained 888 single cells for which we had high-quality RNA-seq data. We computed two FUCCI scores for each cell by individually summing the EGFP (green) and mCherry (red) intensities in a fixed cell area (100 × 100 pixels), correcting for background noise outside the defined cell area, and then taking the log₁₀ transformation of the sum of corrected intensities. In the bottom right scatter plot, we show the FUCCI scores for the 888 high-quality single-cell samples, namely, mCherry and EGFP log₁₀ sum intensities after background noise correction. Finally, we standardized the molecule counts to counts per million (CPM) and transformed the data per gene to a standard normal distribution.