Effect of H3K36me2 on major satellite transcription. Pmi28 cells stably expressing dCas9-Halo-empty or dCas9-Halo-NSD2 and major satellite-specific sgRNA were established. (A) Immunofluorescence. Cells were fixed and stained with JF646 HaloTag ligand (red), H3K36me2-specific antibody (green), and Hoechst 33342 (blue). Confocal images with individual contrast adjustments to compare the localization patterns are shown on the left. Box plots displaying the intensity ratios of H3K36me2 in PCH to the nucleus are shown on the right. P values were calculated by Mann–Whitney U test. See Fig. 1 legend for the details of box plots. (B) RT-qPCR for dCas9-expressing cells. Levels of major satellite transcripts normalized by GAPDH in cells expressing dCas9-Halo-NSD2 relative to those in cells expressing dCas9-Halo-empty are shown (mean of three biological replicates with the standard error and individual data points). P values were calculated by Student’s t test (unpaired, two-tailed). (C) RT-qPCR for KO cells. Levels of major (left) and minor satellite transcripts (right) in WT, NSD2-KO, SMARCA5-KO, and BAZ1B-KO iMEFs are plotted as in B. P values were calculated using the Mann–Whitney U test and Hommel correction to the WT. Scale bar, 10 μm.