Comparative analysis between single-cell RNA-seq and single-molecule RNA FISH indicates that the pyrimidine nucleobase idoxuridine (IdU) globally amplifies transcriptional noise.

Stochastic fluctuations (noise) in transcription generate substantial cell-to-cell variability, but the physiological roles of noise have remained difficult to determine in the absence of generalized noise-modulation approaches. Previous single-cell RNA-sequencing (scRNA-seq) suggested that the pyrimidine-base analog (5’-iodo-2’-deoxyuridine, IdU) could generally amplify noise without substantially altering mean-expression levels but scRNA-seq technical drawbacks potentially obscured the penetrance of IdU-induced transcriptional noise amplification. Here we quantify global-vs.-partial penetrance of IdU-induced noise amplification by assessing scRNA-seq data using numerous normalization algorithms and directly quantifying noise using single-molecule RNA FISH (smFISH) for a panel of genes from across the transcriptome. Alternate scRNA-seq analyses indicate IdU-induced noise amplification for ~90% of genes, and smFISH data verified noise amplification for ~90% of tested genes. Collectively, this analysis indicates which scRNA-seq algorithms are appropriate for quantifying noise and argues that IdU is a globally penetrant noise-enhancer molecule that could enable investigations of the physiological impacts of transcriptional noise.