Fig 1.
Experimental design and global identification of rhythmic transcripts.
(A) Time course RNA sequencing profiles and analyses across 28 hours from T0 to T28 at 4-hour intervals, initiated 24 hours after DEX synchronization. (B) Principal component analysis (PCA) of RNA-seq samples. Points are colored by time. Axes display PC1 and PC2 with the percentage of variance explained. (C) The distribution of the coefficient of variation (CV) of gene expression across three biological replicates at each time point, and the red line indicates the mean CV at T0. Kolmogorov–Smirnov (K-S) test P < 0.001 (T0 v.s. T28, T32, T36, T40, T44, T48). (D) The number of rhythmic genes identified across different analysis window lengths using the meta2d algorithm, based on thresholds of P value < 0.05 or BH-adjusted Q value < 0.05.
Fig 2.
Core clock gene expression in HEK293T cells across the T0-T28.
(A) Time-series expression profiles of 10 core clock genes in HEK293T cells. Expression values represent mean FPKM±SD, n = 3. The BH. Q values calculated from the 24 h and 28 h datasets using the meta2d algorithm are annotated in each panel.
Fig 3.
Circadian features of the HEK293T cell transcriptome.
(A) Proportion of rhythmic and arrhythmic genes among all detected genes. Counts and percentages are shown. (B) Heatmaps displaying rhythmic genes HEK293T cells. Genes are ordered by peak phase in the rhythmic condition. (C) Phase distribution of rhythmic genes across the 24-hour cycle. (D) GO, KEGG and REACTOME pathway enrichment analyses of rhythmic genes. (E) Representative GTPase activator and metal ion binding genes. Expression values represent mean FPKM±SD, n = 3. BH.Q are calculated using the meta2d algorithm, Phases were estimated using sine wave–based least-squares fitting. (F) Pathway terms consistent between PSEA and pathway enrichment analysis.
Fig 4.
Classification of time-dependent and time-independent expression patterns among arrhythmic genes.
(A) Schematic overview of the analytical framework used to classify arrhythmic genes into time-independent and time-dependent categories based on differential expression between adjacent time points from T0 to T28. (B) Heatmaps displaying time independent genes HEK293T cells. (C) GO, KEGG and REACTOME pathway enrichment analyses of time independent genes. (D) The number of overlapping genes between DEG sets. (E) Pathway enrichment analyses of time dependent genes in three DEG group: T0 v.s. T4, T8 v.s. T12, T12 v.s. T16.