Anti h3r8me2a

The regular chromatin preparation was performed as described^{50 (link)}. In brief, cells were cross-linked with 1% formaldehyde solution for 10 min and quenched with 0.125 M glycine. For H3R8me2a ChIP, the cell pellets were resuspended in cold CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM PIPES, pH 6.8) containing Triton X-100 (0.5%) and EGTA (1 mM) before 10 min of fixation in 0.5% Formaldehyde solution. After being quenched by 0.125 M Glycine, cells were spun down, washed, resuspended, lysed, and ultra-sonicated for 25 min with 30 s ultra-sonication at 30 s intervals (Bioruptor pico, Diagenode, Belgium). The resulting fragmented chromatin extract was precleared with Protein A/G beads (ThermoFisher, Beijing, China) and then incubated overnight with antibodies: anti-H3K4me1, anti-H3K4me3, anti-3K27me3, anti-H3K9me3 (Cell Signaling Technology), anti-H3K27ac (Abcam), anti-H3R8me2a (Novus Biologicals), and Normal anti-rabbit IgG (Cell Signaling Technology) as controls. After stringent washes, elution, and reverse cross-linking, DNA was purified using PCR purification kits (QIAGEN, Hilden, Germany). The primers for ChIP-qPCR analyses at the promoters and enhancers are listed in Supplementary Table 2.

The paraffin-embedded tissue sections were used for examination of HE staining. For the IHC staining, specimen’s tissue slides were de-paraffinized, rehydrated, and antigen retrieval. The tissue slides were blocked for at least 1 h at room temperature and then incubated with appropriate primary antibodies overnight at 4 ℃. The antibodies included anti-PRMT2 (1:100; Abcam), anti-CCNB1 (1:200; Santa Cruz Biotechnology), anti-CCND1 (1:100; Absin), anti-CDK4 (1:200; Wanleibio), and anti-H3R8me2a (1:500; Novus Biologicals). After careful washing, the slides were incubated with horseradish peroxidase (HRP) conjugates using DAB detection. For the IHC analysis, we quantitatively scored the tissue sections according to the percentage of positive cells and staining intensity. We assigned the following proportion scores: 1 if 0–25% of the tumor cells showed positive staining, 2 if 26–50% of cells were stained, 3 if 51–75% stained, and 4 if 76–100% stained.

Total RNA isolated using Trizol reagent (Ambion) was subjected to reverse transcription with Superscript Reverse Transcriptase (Invitrogen). RT-qPCR reactions were performed using SYBR Green Master Mix (Thermo) on 7500 Fast real-time PCR system (Applied Biosystems). rPO was used as a reference gene for all qRT-PCR experiments and analyses. The ΔΔCt method was used for quantification analysis. To obtain whole-cell protein extracts or histones, cells were lysed or extracted as previously described^{46 (link)}. Membranes were blotted with the corresponding primary antibodies: anti-PRMT2 (1:1000, LifeSpan BioSciences), anti-STAT3 (1:1000, Abways), anti-p-STAT3 (1:1000, Abways), anti-α-Flag (1:2000, Sigma-Aldrich), anti-PTEN (1:1000, Cell Signaling Technology), anti-AXIN2 (1:500, Absin), anti-Histone H3 (1:4000, Millipore), anti-H3R8me2a (1:1000, Novus Biologicals), anti-H3K4me3 (1:1000, Cell Signaling Technology), anti-H3K27ac (1:1000, Abcam), and anti-GAPDH (1:5000, Abways). After careful washing, bound antibodies were detected with HRP-linked anti-mouse or anti-rabbit IgG (CST), followed by electrochemiluminescence (PerkinElmer) determination. All uncropped WBs can be found in Supplementary Figs. 11 and 12. Primer sequences and information for antibodies are available in Supplementary Table 2.