Extended Data Fig. 6: Characterization of H4Kacme antisera.

a, Chemical structure of the peptide used to generate H4Kacme antisera. Yellow box indicates the H4K5acme residue. b, Peptide dot blots of H4Kacme antisera with synthetic histone peptides. Yellow boxes indicate H4Kacme peptides, and the green box indicates a H3K27acme peptide. c, Coomassie stain of H4Kacme antisera after Protein A and affinity purification and SDS-PAGE. Gels were repeated independently twice with similar results. d, Peptide dot blots of purified H4Kacme antisera with synthetic histone peptides. Yellow boxes indicate H4Kacme peptides, and the green box indicates a H3K27acme peptide. e, Western blot of histones extracted from two biological replicates of HEK-293T cells. f, Western blot of whole nuclear proteins from two biological replicates of HEK293T cells using H4Kacme antisera pre-incubated with indicated competitor peptide. g, Western blots of histones extracted from two biological replicates of HEK293T cells using H4Kacme antisera pre-incubated with indicated competitor peptides. h, Western blots of histones extracted from HEK-293T cells transfected with GFP-H4 plasmids with the indicated genotypes. Blots were repeated independently twice with similar results. i, H4Kacme ChIP-qPCR in HEK293T cells after incubation of H4Kacme antisera with indicated competitor peptide (100 nM). Bar plots show the mean of two biological replicates. j, H4Kacme ChIP-seq read counts in HEK293T cells after incubation of H4Kacme antisera with indicated competitor peptide (100 nM). Box plots show the distribution of mean read counts for each peak from two biological replicates. Distributions were compared using a two-tailed unpaired t-test. The box plots indicate the median (centre line), the third and first quartiles (box limits), 1.5x interquartile range (IQR) above and below the box (whiskers), and the 95% confidence interval (notches). Outlier points are not plotted. *** p < 2.2e⁻¹⁶; NS = not significant. n = 478. k, Genome browser tracks of Kacme and H4Kacme ChIP-seq after incubation of Kacme or H4Kacme antisera with indicated competitor peptide (75 nM for Kacme, 100 nM for H4Kacme). Yellow boxes highlight Kacme competitor peptide tracks.