Extended Data Figure 1. Leading strand bias of H3K9me3 eSPAN is detected by H3K9me3 antibodies from three different sources.
a. The TA skew and average BrdU-IP-ssSeq bias (BrdU bias) around replication origins in mES cells.
b. The TA skew was correlated with BrdU bias. The BrdU bias, reflecting the relative amount of DNA synthesis at leading and lagging strands, was calculated using formula . and represent sequencing reads of Watson and Crick strands, respectively. Spearman’s rank correlation coefficient was shown. Each dot represents a 1 kb bin within the 1,928 initiation zones in mES cells. p < 2.2e-16.
c. Average eSPAN bias of MCM2, a subunit of the CMG replicative helicase, and 7 histone modifications (H4K20me2, H3K36me3, H3K36me2, H3K4me3, H4K12ac, H4K5ac and H4K5/K12ac) in mES cells. Two independent repeats, indicating by blue and red lines, for each eSPAN experiment shown.
d. Raw average H3K9me3 eSPAN bias (ctr) and after normalizing against BrdU bias (no BrdU bias) or TA skew (no TA skew) in mES cells.
e. Average bias of H3K9me3 eSPAN bias generated using three H3K9me3 antibodies from different sources.
f. Immunoblots of H3K9me3 in different amounts of mES cell lysates by three different H3K9me3 antibodies used in e. Recombinant histone H3/H4 (re.) were used as negative controls. * indicates non-specific signals detected by antibody 2 (Ab2, Diagenode) and antibody 3 (Ab3, Active motif) after heavy exposure. Ab1: self-made in the laboratory and used in this study. n = 3.
g. Genome-wide correlations of ENCODE H3K9me3 ChIP-seq with H3K9me3 CUT&Tag signals generated with three different antibodies Ab1, Ab2 and Ab3, with a window size of 20 kb. Note that Ab1 showed the strongest correlation with published ChIP-seq datasets, consistent with the immunoblotting results. These differences in performances of the three H3K9me3 antibodies likely contribute to the different H3K9me3 eSPAN biases observed in e.
h. A snapshot of ENCODE H3K9me3 ChIP-seq signals and two repeats of H3K9me3 CUT&Tag signals generated by Ab1 H3K9me3 antibodies at the indicated mouse Chr14 region.
For gel source data, see Supplementary Figure 1.