Ab13604

Three biological replicate ChIP‐seq experiments were conducted for the specific detection of AML1‐bound genomic regions on THAP10 and miR‐383 promoters according to standard procedures with several modifications. Briefly, cross‐linked chromatin from approximately 5–10 × 10⁷ SKNO‐1, SKNO‐1‐siA/E, U937 and U937‐A/E cells was prepared and fragmented to an average size of approximately 200 bp by 30–40 cycles of sonication (30 s each) in 15 ml tubes using the Bioruptor UCD‐200 sonicator (Diagenode). For immunoprecipitation, AML1 (sc‐8563, Santa Cruz Biotechnology), ETO (sc‐9737, Santa Cruz Biotechnology), HDAC1 (ab7028, Abcam), DNMT1 (ab13537, Abcam), DNMT3a (ab13888, Abcam), DNMT3b (ab13604, Abcam) and p300 (ab14984, Abcam) antibodies were added to 12 ml of diluted, fragmented chromatin. Nonimmunized rabbit serum served as a control. ChIP using the normal mouse IgG (Abcam) antibody was performed on naked and sonicated DNA extracted from the same cell samples and assayed using the EZ‐ChIP™ Chromatin Immunoprecipitation kit (Millipore) as per the manufacturer's instructions. Genomic THAP10 and pre‐miR‐383 upstream regions close to the putative AML1‐binding site were amplified. Primer sequences are shown in Appendix Table S7. GAPDH served as a control for nonspecific precipitated sequences.

Cells were washed twice in PBS, collected, and lysed in cell lysis buffer on ice for 30 min and then sonicated for 1 min at 60 of amplitude with 2 s intervals. After centrifugation at 10,000g, 4 °C for 10 min, supernatant was transferred into new tubes. The concentration of the protein sample was measured by bicinchoninic acid, and then protein samples were boiled in SDS Sample Buffer at 99 °C for 10 min. 20 μg or 40 μg (for Tcstv1/3 and Zscan4) total proteins of each cell extracts were resolved by 10% or 12% (for Tcstv1/3) Bis-Tris SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF; Millipore). Nonspecific binding was blocked by incubation in 5% skim milk in TBST at room temperature for 2 h. Blots were then probed with primary antibodies, Tcstv1/3 (custom-made), Zscan4 (AB4340; Millipore), H3K9me3(ab8898; Abcam), H3K9Ac (04-1003; Millipore), H3Ac (06-599; Millipore), H3 (ab1791; Abcam), Dnmt3a (ab13888; Abcam), Dnmt3b (ab13604; Abcam) and β-actin (P30002; Abmart) by overnight incubation at 4 °C in 5% skim milk in TBST. Immunoreactive bands were then probed for 2 h at room temperature with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-Rabbit IgG-HRP (GE Healthcare, NA934V), or goat anti-Mouse IgG (H + L)/HRP (ZSGB-BIO, ZB-2305). Protein bands were detected by Chemiluminescent HRP substrate (Millipore, WBKLS0500).

ChIP-qPCR analysis was performed as described previously [72 (link)], with slight modification. Briefly, 5 × 10⁷ cells were fixed with 1% paraformaldehyde, lysed, and sonicated to achieve the majority of DNA fragments with 100–1000 bp. DNA fragments were then enriched by immunoprecipitation with 5 μg H3K9me3 antibody (ab8898, Abcam), 7 μg H3K9Ac antibody (ab4441, Abcam), 5 μg Dnmt3b antibody (ab13604, Abcam), 5 μg H3K9me2 antibody (ab1220, Abcam) or 5 μg H3K27me3 (ab6002, Abcam). The eluted protein:DNA complex was reverse-crosslinked at 65 °C overnight. DNA was recovered after proteinase and RNase A treatment. Real-time PCR was performed to compare the histone modification at the Fst promoter region using primers provided in S5 Table. Normal rabbit IgG (#2729S, Cell Signaling) or Mouse (G3A1) mAb IgG1 Isotype Control (5415S, Cell Signaling) served as negative control.