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. 2021 Mar 8;10:e62161. doi: 10.7554/eLife.62161

Figure 1. Moderate averaging represents a valid approach for origin recognition complex/minichromosome maintenance complex (ORC/MCM) chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis.

(a) Sequencing profile visualization in UCSC Genome Browser (hg19) at the Mcm4/PRKDC origin after reads per genomic content normalization: two samples of Orc2 and Orc3, and three samples of Mcm3 and Mcm7, are plotted against the input in three replicates. The profiles are shown in a 10 kb window (chr8: 48,868,314–48,878,313); the mapped position of the origin is indicated as green line. (b) The profile of ORC/MCM ChIP-seq after 1 kb binning at the same locus. The reads of replicates were summed and normalized by the total genome-wide ChIP read frequency followed by input division. Y-axis represents the resulting relative read frequency. (c) Correlation plot between Orc2 and Orc3 relative read frequencies in 1 kb bins. (d) Correlation plot between Mcm3 and Mcm7 relative read frequencies in 1 kb bins. (e) Heatmap of Pearson correlation coefficients r between all ChIP relative read frequencies in 1 kb bins. Column and line order were determined by complete linkage hierarchical clustering using the correlation distance (d = 1 r). Refer to Figure 1—figure supplement 3 for data representation without input division.

Figure 1.

Figure 1—figure supplement 1. Experimental validation of cell cycle fractionation and origin recognition complex and minichromosome maintenance complex (ORC/MCM) chromatin immunoprecipitation followed by sequencing quality.

Figure 1—figure supplement 1.

(a) Example DNA content (propidium iodide) staining followed by FACS of logarithmically growing Raji (top) cells and after cell cycle fractionation by centrifugal elutriation (increasing counter flow rates indicated above each profile in ml/min). (b) Western blot analyses of the single fractions detecting cyclin A (S/G2), cyclin B (G2/M), H3S10P (M), and GAPDH. (c) qPCR validation of Orc2, Orc3, Mcm3, and Mcm7 enrichment at the Epstein–Barr virus latent origin oriP dyad symmetry element. Representation in % input. Isotype IgG was used as control. *p<0.05, **p<0.01.
Figure 1—figure supplement 2. The input sequencing control is differentially represented in regions of biological function.

Figure 1—figure supplement 2.

(a) Heatmaps of Pearson correlation coefficients r between all chromatin immunoprecipitation relative read frequencies at different bin sizes: 100 bp, 1 kb, 10 kb, and 100 kb. (b–d) Boxplot of normalized read frequencies in relation to (b) DNase hypersensitivity. DNase hypersensitive (HS) clusters were obtained from 125 cell lines in ENCODE, only HS sites larger 1 kb were considered. (c) Transcription: transcription start sites and gene bodies of active (transcripts per kilobase per million [TPM] >3) and inactive (TPM <3) gene. (d) Early- or late-replication timing domains. The dashed gray horizontal line indicates read frequency 1.0 for orientation. Boxplot represents the mean (circle), median (thick line), first and third quartile (box), and first and ninth decile (whiskers) of the normalized read frequencies, without representing outliers. Statistics were performed using one-sided t-test. ***p<0.001.
Figure 1—figure supplement 3. Origin recognition complex/minichromosome maintenance complex (ORC/MCM) enrichments at the MCM4/PRKDC origin persists without input normalization.

Figure 1—figure supplement 3.

(a) The profile of ORC/MCM chromatin immunoprecipitation followed by sequencing (ChIP-seq) after 1 kb binning in the same 10 kb window as Figure 1b (chr8: 48,868,314–48,878,313). The reads of replicates were summed and normalized by the total genome-wide ChIP read frequency. Y-axis represents the resulting normalized read frequency. (b) Correlation plot between Orc2 and Orc3 normalized read frequencies in 1 kb bins. (c) Correlation plot between Mcm3- and Mcm7-normalized read frequencies in 1 kb bins. (d) Heatmap of Pearson correlation coefficients r between all ChIP-normalized read frequencies including input in 1 kb bins. Column and line order were determined by complete linkage hierarchical clustering using the correlation distance (d = 1 r). (e) ORC relative read frequencies at Orc2 peaks (>1 kb) retrieved from Miotto et al., 2016. (f, g) ORC/MCM binding is confirmed at DNase hypersensitive (HS) sites. (f) Mean input-normalized ORC/MCM relative read frequencies (±2 × SEM) in relation to DNase hypersensitivity. (g) ORC/MCM-normalized read frequencies without input division (±2 × SEM) in relation to DNase hypersensitivity. Only HS sites larger than 1 kb were considered. Statistics were performed using one-sided t-test. ***p<0.001.