Ab8898

Protein extracts were derived from adult fly heads, lysed in sample buffer or Urea Buffer (150 mM NaCl, 10 mM Tris-HCl pH8, 0,5 mM EDTA, 10% glycerol, 5 mM EGTA, 50 mM NaF, 4 M urea, 5 mM DTT, Protease Inhibitor Cocktail (PIC) (Roche), fractionated by SDS-PAGE and transferred to nitrocellulose membrane. Primary antibodies were: anti-TBPH rabbit (1:1000; homemade [18 (link)]); anti-Actin goat (1:1000; Santa Cruz, sc-1616); anti-Vibrator rabbit (1:5000; also named Giotto [54 (link)]); anti-H3K9me2 mouse (1:400; Abcam ab1220), anti-H3K9me3 rabbit (1:1000; Abcam ab8898); anti-Tubulin mouse (1:5000; Sigma, T-5168); anti-HA HRP (1:1000; Santa Cruz sc7392); anti-Su(var)3-9 rat (1:50; [33 (link)]). As a secondary antibody, we used the appropriate HRP-conjugated antibody (GE Healthcare) diluted 1:5000 in PBS-Tween 0.1%. Membranes were incubated 5 min with ECL substrate (#1705062 and #1705060, Bio-Rad) and the HRP-ECL reaction was revealed using the ChemiDoc^TM XRS gel imaging system (Bio-Rad). Band intensity quantification was performed using the gel analyzer tool in Fiji/ImageJ software.

Basically, cultured oocytes were fixed in 2% paraformaldehyde (PFA) in PBS containing 0.1% Triton X-100 for 30 min. Only for microtubule staining, cultured oocytes were preincubated with ice-cold M2 for 10 min and proceeded to fixation as described above. After permeabilization with 0.1% Triton X-100 in PBS overnight at 4°C, oocytes were incubated with primary antibodies overnight at 4°C. Following three washes with PBS containing 0.1% polyvinyl alcohol (PVA), Alexa-Fluor-labelled secondary antibodies (Invitrogen) were used for the detection of signals and DNA was counterstained with 14.3 μM 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, Invitrogen). The following primary antibodies were used: anti-REC8 (a kind gift from Jibak Lee, Kobe University, Japan; 1:100), anti-centromere protein (1:100), anti-SGOL2, anti-Meikin (kind gifts from Yoshinori Watanabe, University of Tokyo, Japan; 1:100 and 1:100, respectively), anti-TOPOII (Abcam, #ab109524, 1:100), anti-histone H3 tri-methylated at lysine 9 (H3K9me3; Abcam, #ab8898, 1:250-500) and anti-alpha Tubulin (Sigma, #T9026, 1:250-500). Samples were mounted on a slide with Vectashield (Vector Laboratories), covered with a No.1.5H (170 μm ± 5 μm) coverslip (Marienfield) and were imaged using a confocal laser scanning microscopy.

Cortex homogenate was used for H3K27me3 ChIP. As for H3K9me3, Nuclei were extracted from the cortex of male offspring and neuronal (NeuN +) nuclei were enriched by FANS sorting using MoFlo Astrios EQ cell sorter (Beckman Coulter). Native ChIP was performed as described (Jiang et al. 2017 (link)). Briefly, the chromatin was digested with MNase at 28 ℃ for 10 min to obtain mononucleosomes and incubated with anti-H3K9me3 (Abcam AB8898) or anti-H3K27me3 (Millipore, 07–449) antibody at 4 ℃ overnight. The immunoprecipitated complexes were captured by protein A/G magnetic beads (Thermo Scientific, 88803) and washed with low-salt buffer, high-salt buffer, Lithium Chloride buffer and TE buffer. ChIP DNA was then eluted in elution buffer and incubated with RNase A, followed by proteinase K incubation. Finally, ChIP DNA was purified using SPRI magnetic beads (Beckman, B23318).
For ChIP DNA library preparation, End-repairing (Lucigen Corporation, ER0720) and A-tailing (Lucigen Corporation, KL11101K) was performed and then ChIP DNA was ligated (Lucigen Corporation, LK0750H) with Y-adaptor (Vazyme, N802) and subjected to PCR amplification (Vazyme, N618-01). Library DNA was size-selected with SPRI beads and sent to GENEWIZ,China, for deep sequencing with Novaseq set paired-end, 150 bp (PE150).

Snap‐frozen mouse liver (100 mg) from young and old wild‐type mice was used to prepare chromatin. ChIP and H3K14ac ChIP‐Seq were performed as described previously (Whitton et al., 2018). For Setdb1 and Kap1, a few modifications were incorporated. Particularly, sonication, using Diagenode Bioruptor Pico, was reduced to 11 cycles (30 s pulse on, 30 s pulse off) and libraries were sequenced using Illumina NextSeq 500 following the manufacturer's protocols. Rabbit polyclonal antibodies specific to KAP1 (Abcam, ab10483), SETDB1 (Proteintech, 11231–1‐AP), and anti‐acetyl‐histone‐H3 specific to Lys‐14 (Millipore, 07–353) were used for immunoprecipitation.
For sequential ChIP, the first immunoprecipitation was performed as described for ChIP (Whitton et al., 2018) with some modifications. After the washes, samples were eluted in 100 µl 1% SDS and 10 mM fresh dithiothreitol (DTT) twice for a total elution volume of 200 µl. After each elution, the samples were rotated at 37ºC for 15 min. After the second elution, samples were diluted in TE to 1.5 ml. Then, the second antibody was added and the second ChIP was performed as described previously (Whitton et al., 2018). Rabbit antihistone H3 (trimethyl K9) antibody (ab8898) and anti‐acetyl‐histone‐H3 specific to Lys‐14 (Millipore, 07–353) were used for immunoprecipitation.

For ChIP assays, CD14+ cells (MOs) treated with IL-4/GM-CSF for 0 and 2 days were crosslinked with 1 % formaldehyde and subjected to immunoprecipitation after sonication. ChIP experiments were performed using a low cell ChIP kit (Diagenode SA, Seraing, Belgium). They were analysed by real-time quantitative PCR. Data are represented as the ratio of the bound fraction to the input for each specific factor. We used an antibody against STAT6 (Santa Cruz, sc-981x), and histone marks H3K9me3 (Abcam, ab8898) and H3K27me3 (Millipore 07-449). Human IgG was used as a negative control. Primer sequences were designed to contain predicted or known TF binding (from TRANSFAC or ChIPseq data) as close as possible to the CpG undergoing methylation changes. Primer sequences are shown in Additional file 11. Three biological replicates of the experiments were performed.

Whole cell extract or, for histone analysis, nuclear extract was run on either 10 or 15% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels at 150 V until separated and transferred at 4 °C for either 1 h at 80 V (histones) or overnight at 20 V (Phf8) onto a PVDF membrane. Membranes were blocked in 5% milk PBS-T (1 × PBS, 0.1% Tween-20) for 30 min at RT then incubated with primary antibodies for 1 h in block at RT. Membranes were washed in PBS-T, and incubated in the appropriate horseradish-peroxidase-conjugated secondary for 45 min at RT. Membranes were then washed again and visualized using ECL reagents (Pierce). Primary antibodys: Phf8 Abcam ab36068 1:400, H3 Abcam ab1791 1:5,000, H3K9me1 Abcam ab8896 1:500, H3K9me2 Abcam ab1220 1:200, H3K9me3 Abcam ab8898 1:1,000, H3K27me2 Millipore 07-452 1:2,000, H4 Abcam ab17036 1:1,000, H4K20me1 Abcam ab9051 1:1,000, beta Actin-HRP Abcam ab20272 1:8,000. All uncropped blots can be found in Supplementary Fig. 11.

Chromatin from cells was fragmented to a size range of 200–500 bases with a Sonicator. Solubilized chromatin was immune‐precipitated with antibody against H3K4me2 (Abcam, ab7766, 1:1,000), H3K4me3 (Abcam, ab8580, 1:1,000), H3K9me2 (Abcam, ab1220, 1:1,000), H3K9me3 (Abcam, ab8898, 1:1,000), H3K27me3 (Millipore, 17‐622, 1:2,000), and H3K36me3 (Abcam, ab9050, 1:1,000). Antibody–chromatin complexes were pulled down with protein A/G (Invitrogen), washed, and then eluted. After cross‐link reversal and proteinase K treatment, immune‐precipitated DNA was extracted with phenol–chloroform, ethanol precipitated, and treated with RNase. ChIP DNA was quantified using PicoGreen. For ChIP‐qPCR, primer sequences are listed in Dataset EV6. qPCR was performed on the CFX96 Real‐Time System (Bio‐Rad) with SsoAdvanced SYBR Green Supermix (Bio‐Rad).