Anti trimethyl histone h3 lys27

Protein samples were extracted from nuclear and cytoplasmic fractions according to protocol by Gagnon et al.,^{18 (link)} and with TRI reagent (Molecular Research Center) according to the manufacturer’s instructions. For western blot, equal volumes of extracted protein were loaded on precast gels (Mini-Protean TGX Stain-free Precast Gel, 4–20%, Bio-Rad) and transferred to nitrocellulose membranes (Trans-Blot Turbo Bio-Rad Midi, 0.2 μm nitrocellulose, Bio-Rad). The membranes were blocked with 5% milk for 1.5 hours at room temperature, washed with TBST (0.15 M sodium chloride, 0.050 m TRIS-HCl buffer; 0.05% Tween 20; pH 7.6) and incubated with antibodies against a known nuclear protein (anti-trimethyl-histone H3 (Lys27), Millipore, 1:2,500) and a cytoplasmic protein (anti-β-tubulin, Sigma Aldrich, 1:1,000) overnight at 4 °C. The membranes were washed with TBST and incubated with secondary antibodies (goat anti-rabbit IgG (H + L), HRP-conjugated, Invitrogen; anti-mouse IgG, HRP-conjugated, R&D systems; both 1:5,000) for 1 hour at room temperature. Membranes were analyzed using ECL Plus Western Blotting Substrate (Pierce) and imaged with the ChemiDoc Imaging System (Bio-Rad).

Chromatin was cross-linked for 12 min with 1% formaldehyde (Sigma) and glycine was then added to a final concentration of 0.125 M for 5 min. After washing and collecting the cells, samples were incubated in nuclei lysis buffer (50 mM Tris-HCl pH 8.1, 1% SDS, 10 mM EDTA) on ice for 30 min. Sonication to obtain chromatin fragments of around 200–300 bp was performed using a Bioruptor UCD-200 sonicator (Diagenode). Chromatin extracts were immunoprecipitated overnight on a rotating platform at 4 °C with 3 μg the following antibodies: anti-MyoD (Santa Cruz, sc-760) and anti-trimethyl-histone H3 (Lys27) (Millipore 07-449). Normal rabbit IgG was used as negative control (mock). Immunoprecipitated chromatin was conjugated with G-protein magnetic Beads (Invitrogen). After extensive washing, bound DNA fragments were eluted and analysed by quantitative PCR using the SYBR Green Master Mix (Applied Biosystems). Primers sequences are indicated in Supplementary Table 2.

Cells were permeabilized with 1% Triton X-100 in 1x PHEM buffer (10 mM EGTA, 25 mM HEPES, 2 mM MgCl₂, 60 mM PIPES (pH 6.9)) for 10 min and then fixed with 2% Paraformaldehyde for 10 min. The cells were blocked in 3% BSA in TBSTEM buffer (10 mM EGTA, 2 mM MgCl₂, 0.15 M NaCl, 10 mM Tris, 1% Tween 20 (pH 7.4)) for an hour. After blocking, Anti-trimethyl-Histone H3 (Lys27) (07-449, Millipore), anti-trimethyl-Histone H3 (Lys9) (07-442, Millipore) or anti-ubiquityl-Histone H2A (Lys119) (8240T, Cell Signaling Technology) was used as the primary antibody at a dilution of 1:200 and incubated overnight at 4°C. After washing twice with 3% BSA in TBSTEM, the cells were incubated for an hour with goat anti-rabbit Alexa Fluor 546 Secondary antibody (A-11071, Invitrogen) at a dilution of 1:4,000. The cells were washed twice with 1x PBS, spread on glass slides and mounted with ProLong Glass Antifade Mountant (P36980, Invitrogen), formulated with the blue DNA stain NucBlue.

ChIP was performed with GC cells following a protocol from the Myers’ laboratory (http://hudsonalpha.org/myers-lab/protocols, accessed on 15 June 2021) with modifications. Briefly, the cells were fixed with 1% formaldehyde, lysed, and sonicated using a Covaris M220 (Covaris, Woburn, MA, USA). For ChIP-PCR analysis, the sonicated lysates were used by dividing the same amount into three tubes and a 10% input. Each of the samples were immunoprecipitated with normal Rabbit IgG (2 μg; Millipore; 12-370), anti-trimethyl-Histone H3 (Lys4) (2 μg; Milipore; 07-473) or anti-trimethyl-Histone H3 (Lys27) (5 μg, Millipore; 07-449) using 50 μL Dynabeads coupled with protein A and protein G (Invitrogen), respectively. Immunoprecipitated DNA was recovered using the QIAquick PCR Purification kit (Qiagen) and used to amplify three regions around TSS of SSTR2. The PCR products were analyzed on 1.5% agarose gels stained with GelRed (biotium). The primer sequences for RT-PCR are listed in Supplemental Table S1.

Cells were collected during sexual development and prepared for immunostaining according to standard protocol [31] (link). Anti-trimethyl-Histone H3 (Lys9) antibody (07-442, Millipore) and Anti-trimethyl-Histone H3 (Lys27) (07-449, Millipore) at 1∶100 dilution were used. A FLUOVIEW FV1000 (Olympus) system with PLAPON 60× O SC NA 1.40 objective was used for imaging capture.