Fig. 2. Transcription intensification is not detected using standard RNA-seq.

A Distribution of transcript levels, expression fold changes (Log₂), and numbers of differentially expressed (DE) genes (q-value < 0.01 for a |Log2 FC | > 0.5) using a standard RNA-seq analysis of 5 biological replicates comparing cotyledons dissected from dark- or light-grown seedlings. The p-value was calculated from a two-sided Welch’s t test. B RNA Pol II S2P and H2Bub chromatin enrichment at differentially expressed genes analyzed in A, genes displaying similar transcript levels in dark and light (Similar: q-value < 0.01 for |Log2 FC | ≤ 0.5), and genes neither up nor downregulated (non-DEGs) in standard RNA-seq analysis of dissected cotyledons. C RNA Pol II S2P and H2Bub meta-profiles over all the genes displaying similar transcript levels in dark and light as defined in (B). The p-values are calculated from a two-sided Wilcoxon Rank Sum test. Three independent biological replicates were analyzed for RNA Pol II S2P, and two for H2Bub. Source data are provided as a Source Data file. D Experimental setup for introducing Drosophila S2 cells as an exogenous spike-in reference (RNA-Rx). As a proof-of-concept experiment, 5000 S2 cells were mixed with either 100 or 200 dissected cotyledons prior to RNA extraction. Created in BioRender. Richet-Bourbousse, C. (2025) https://BioRender.com/rrgd2r4E RT-qPCR analysis of mRNAs extracted from 100 or 200 dissected cotyledons, both including the same number of Drosophila cells as a spike-in. The ratio of RNA levels between the two sample types was determined either by normalizing to Arabidopsis reference genes (AT3G02065, PP2A, and AT2G41020) or to Drosophila genes (RpL21, cg25c, vkg, B4, and pum). Error bars indicate the standard deviation between two technical replicates. F Distribution of expression changes (Log₂ FC). RNA sequencing reads were analyzed by normalizing solely to library size (Standard) or using the reads mapping to Drosophila genes (Spike-in normalization). The analysis uses four independent biological replicates. Source data are provided as a Source Data file.