Figure 2. Labeling zygotic RNAs in late 2C embryos using 4sU.

(A) Schematic diagram describing the zygotic RNA labeling experiment in late 2C embryos. Briefly, embryos grew in medium containing 4sU. Newly transcribed zygotic RNAs after ZGA incorporated 4sU and were labeled with T->C changes when alkylated by Iodoacetamide (IAA) and converted to cDNAs during RT.

(B) Observed conversion rate of all mutation types in no-4sU control (n=7, top) and 4sU labeled (n=12, bottom) embryos on the sense strand within genes. Total RNAs were divided into three groups based on their allelic origins: Paternal, maternal and neutral which were undistinguishable between two alleles.

(C) Examples in 4sU-seq showing RNA labelling efficiency for zygotic genes with high transcriptional potential at the onset of ZGA, as indicated by the heatmap. Genes were analyzed both in bulk (Total) and individually. Selected genes are indicated by the black arrows. A maximum of one experimental replicate may be removed due to the strong deviation from others.

(D) Examples of RNA labelling efficiency for possible maternally deposited genes with expected low transcriptional potentials after ZGA. Analyses were the same as (C).

(E) 4sU-seq: X:A dosage compensation (X:A ratio) in male and female embryos using 4sU-labeled zygotic polyA transcripts at the late 2C stage.