Glycogen

Cells (5 × 10⁶ cells per assay) were cross-linked with 1% formaldehyde (Sigma; F8775), quenched with 0.125 M glycine, and then sonicated using a Bioruptor UCD-300 (Diagenode) to yield chromatin fragments. Then 100 μg of lysate was immunoprecipitated with anti-IgG (negative control), anti-PRMT5 (Sigma; P0493), anti-H4R3me2s (Abcam: ab5823), or anti-H3R8me2s (Abcam; ab272149) antibodies immobilized on protein A-Sepharose beads (Sigma; P3391). The immunoprecipitated complexes were washed sequentially with washing buffer (10 mM Tris–HCl, 500 mM NaCl, 1% Trition X-100, 0.1% SDS, 0.5% Na-deoxycholate). After digestion with proteinase K (Beyotime; ST532), the DNA fragments were extracted with phenol:chloroform:isoamyl alcohol (25:24:1 v:v:v) and precipitated with 100% ethanol at -20 °C in the presence of 0.3 M sodium acetate (pH 5.2) and 2 µg of glycogen (Beyotime; D0812). Finally, the glycogen-protein pellet was suspended in H₂O and the purified DNA was subjected to real-time PCR analyses. The ChIP primer sequences are listed in Supplementary Table S4.

To prepare chromatin, cells were collected and cross-linked with 1% formaldehyde for 10 min at room temperature. The crosslinking reaction was stopped by adding glycine (2 M) and the chromatin was sonicated to generate 200 ~ 500 bp DNA fragments. Immunoprecipitation was performed by adding either anti-PRMT5 (Sigma, P0493), anti-H4R3me2s (Abcam, ab5823), anti-H3R8me2s (Abcam, ab272149), anti-EZH2 (Abcam, ab191250), H3K27me3 (Abcam, ab8898), anti-IgG (Abcam, ab172730) and Protein G agarose beads (salmon sperm DNA pre-treated) to each sample and incubated overnight at 4 °C. Following extensive washing with, the complexes were decrosslinked at 65 °C and treated with Proteinase K (Roche). The eluted DNA was precipitated with ethanol at -20 °C in the presence of Glycogen (20 mg/mL, Beyotime, D0812) as a carrier, washed twice with 70% ethanol, and then subjected to real-time PCR using primers specific to the target gene promoters, which were listed in Table S3.