Rabbit anti h3k27me3

Proteins (20–40 µg) were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with rabbit anti-HA tag (1:1000, C29F4, Cell Signaling, Danvers, MA), rabbit anti-KDM6B (1:1000, GTX124222, GeneTex, Irvine, CA), mouse anti-NEFM (1:200, NF-09, sc-51683, Santa Cruz Biotechnology, Dallas, TX) or mouse anti-α-tubulin (1:5000, B-5-1-2, Sigma-Aldrich). Histones were extracted using the EpiQuik total histone extraction kit (EpiGentek, Farmingdale, NY) and analyzed by immunoblotting using mouse anti-histone H3 (1:1000, 05-499, Millipore, Burlington, MA), rabbit anti-H3K27me3 (1:1000, 07-449, Millipore) or rabbit anti-H3K4me3 (1:1000, ab8895, Abcam, Cambridge, MA). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies. Proteins were visualized using a Clarity Western ECL kit (#1705061, Bio-RAD, Hercules, CA). For visualization using the Odyssey system (LI-COR, Lincoln, NE), goat anti-mouse IRDye 800 or 680 and anti-rabbit IRDye 800 or 680 from LI-COR were used as secondary antibodies.

Cryosections were blocked with 4% HIFCS in THZT buffer (50 mM Tris-HCL, pH 7.6; 0.5 M NaCl; 0.5% Triton X-100) and incubated with a primary antibody; Th [Rabbit-TH (Pelfreeze 1:1,000), Sheep-TH (Millipore AB1542, 1:750)], EYFP [Chicken-GFP (Abcam, 1:500)], H3K27me3 [rabbit-anti-H3K27me3 (Millipore, 17-622 1:2,000)] or Serotonin [rabbit-anti-Serotonin (Immunostar, 1:500)], in THZT buffer overnight at room temperature. The following day the slides were washed and incubated for 2 h at room temperature with secondary Alexafluor antibody [anti-rabbit, anti-sheep and anti-chicken (Invitrogen, 1:1,000)] in Tris-Buffer saline (TBS). After immunostaining nuclei were stained with DAPI (1:3,000), embedded in Fluorsave (Calbiogen) and analyzed with the use of a fluorescent microscope (Leica). All washes were done in TBS and double staining was performed sequentially. The antibody against H3K27me3 required antigen retrieval, which was executed as follows: slides were incubated 10 min in PFA after which they were submerged in 0.1 M citrate buffer pH 6.0 for 3 min at 1,000 Watts followed by 9 min at 200 Watts. Slides were left to cool down, after which protocol was followed as described above. Per embryonic stage two sets, En1Cre/+; Ezh2 +/+ vs. En1Cre/+; Ezh2 L/L, were analyzed. Wnt1Cre driven mutants, En1 mutants and En1;Ezh2 double mutants were performed on one set of embryos.

CUT&RUN analysis was performed with approximately 2 × 10⁵ naive ESCs for each library. Bead-bound cells were incubated overnight at 4 ^oC with Rabbit anti-H3K4me3 (Millipore, #07-473) or Rabbit anti-H3K27me3 (Millipore, #07-449) antibody diluted in the antibody buffer (EDTA/Digitonin/wash buffer, EMD Millipore, #300410) (1 µg/500 µl). The beads were washed three times and incubated with 200 µl pA-MNase (0.7 ng/µl) for 1 h at 4 ^oC. 2 mM of CaCl₂ was added to activate MNase for 30 min on ice. The reaction was quenched by 2× stop buffer containing 340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin, 50 µg/ml RNase A (QIAGEN, #19101), 50 µg/ml glycogen (Roche, #10901393001) and 2 pg/ml Drosophila spike-in DNA at 37 ^oC for 10 min. The soluble chromatins were converted to libraries using the NEBNext Ultra^TM II DNA library Prep kit (NEB, #E7645L). Libraries were subject to pair-ed sequencing on the Illumina NovaSeq 6000 instrument at the University of Michigan core facility.

The following primary antibodies were used: Mouse anti-OCT3/4 (Santa Cruz Biotechnology, Clone C-10, #sc-5279, 1:50), Rabbit anti-CDX2 (BioGenex, Clone EP25, #NU777-5UC, 1:100), Mouse anti-CDX2 (BioGenex, Clone CDX2-88, #MU392A-5UC, 1:100), Rabbit anti-EOMES (Abcam, #ab23345, 1:100), Mouse anti-SMA (SIGMA ALDRICH, Clone 1A4, #A2547, 1:200), Mouse anti-TUJ1 (Biolegend, Clone TUJ1, #801201, 1:200), Goat anti-GATA6 (R&D Systems, #AF1700, 1:100), Mouse anti-5mC (Millipore, Clone 33D3, #MABE146, 1:200), Rabbit anti-H3K27me3 (Millipore, #07-449, 1:100) and Rabbit anti-H3K9me3 (Abcam, #ab8898, 1:100). The secondary antibodies used were: Goat anti-Mouse IgG FITC-conjugated (Thermo Fisher Scientific, #62-6511, 1:50), Goat anti-Mouse IgG Alexa Fluor^R 555 (Abcam, #ab150114, 1:1000), Donkey anti-Goat IgG Alexa Fluor^TM 488 (Thermo Fisher Scientific, #A11055, 1:1000), Donkey anti-Mouse IgG Alexa Fluor^TM 555 (Thermo Fisher Scientific, #A31570, 1:1000), Donkey anti-Rabbit IgG Alexa Fluor^TM 555 (Thermo Fisher Scientific, #A31572, 1:1000), and Donkey anti-Rabbit IgG Alexa Fluor^TM 488 (Thermo Fisher Scientific, #A21206, 1:1000).

Chromatin immunoprecipitation (ChIP) was performed on native chromatin extracted from 2 × 10⁷ ES or 1 × 10⁷ TS cells using standard protocols (40 (link)). Nuclei were purified on a sucrose gradient and chromatin digested with 60 U/ml Micrococcal Nuclease (Affymetrix). Lysates were pre-cleared with Protein G Sepharose beads (GE Healthcare) and incubated with 4 μg of either rabbit anti-H3K9me3 (Abcam ab8898) or rabbit anti-H3K27me3 (Millipore 07–449) at 4°C overnight. Chromatin was immunoprecipitated with Protein G Sepharose beads at 4°C for 4 h. Mock ChIPs were performed in parallel with an isotype-matched control IgG or with beads alone. Eluted DNA from bound and input fractions was subjected to quantitative polymerase chain reaction (PCR) analysis with primer sets specific to genomic promoter regions. Enrichment values were expressed as bound:input ratios and normalized against the corresponding mock values. All ChIPs were performed on at least three biological replicates and compared by T-test. All primers are given in the Supplementary Material.

Cells were fixed as described in [24 (link)] with some modifications. Briefly, cells were collected in centrifuge tubes and fixed for 30 minutes in Solution I (10 mM EGTA, 25 mM HEPES, 2 mM MgCl2, 60 mM PIPES pH 6.9 (PHEM 1X); formaldehyde 1% (Sigma-Aldrich), Triton X-100 2.5%, Sucrose 4%), and for 10 minutes in solution II (PHEM 1X, formaldehyde 4%, Triton X-100 1.2%, Sucrose 4%). Following blocking in 3% bovine serum albumin (Sigma-Aldrich) supplemented Tris buffered saline (10 mM Tris pH 7.4, 0.15 M NaCl) -Tween 20 0.1% (TBST) for 10 minutes, fixed cells were incubated overnight at room temperature with primary antibodies: rabbit anti-H3K9me3 (07–442, Millipore; 1:200), rabbit anti-H3K27me3 (07–449, Millipore; 1:500). After two washes in TBST 3% BSA, cells were labeled with Alexa Fluor 568-conjugated goat anti-rabbit IgG (Invitrogen, catalog number #A-11036, 1:500) for 1 h, stained with 1 μg/mL Hoechst for 5–10 minutes, washed in TBST and finally mounted in Citifluor AF2 glycerol solution (Citifluor Ltd, London).