Histone H3K4me3 antibody (mAb)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.	-It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.	-Once in solution, store 1M DTT at -20°C.

Upstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.
Protocol tips
-It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.
Downstream tips
-Once in solution, store 1M DTT at -20°C.

Publication protocol

ChIP experiments were performed as described in the past36. In brief, for analysis of histone marks we fixed the cells with 1% formaldehyde for 10 min at 25° C and lysed them by the addition of Nuclei Incubation Buffer (15 mM Tris pH 7.5, 60 mM KCl, 150 mM NaCl, 15 mM MgCl2, 1 mM CaCl2, 250 mM Sucrose, 0.3% NP-40) and incubation at 4° C for 10’. The nuclei were washed once with Digest buffer (10 mM NaCl, 10 mM Tris pH 7.5, 3 mM MgCl2, 1 mM Cacl2) and we used micrococcal nuclease (USB) in digest buffer to generate mononucleosomal particles. The reaction was stopped with the addition of EDTA (20 mM). The nuclei were lysed in “nuclei lysis” buffer (50 mM Tris-HCl (pH 8.0), 10mM EDTA (pH 8.0) and 1% SDS) followed by sonication using bioruptor (Diagenode) and chromatin was precleared by the addition of nine volumes of “IP dilution” buffer” (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA (pH 8.0), 16.7 mM Tris-HCl pH 8.0 and 167 mM NaCl) and magnetic Dynal beads. 1% of the chromatin was kept as input. We coupled 2.5 mg antibody with 25 mg antibody for 4 h in reaction buffer and the complex was added to precleared chromatin (equivalent of 105–106 cells, depending on the antibody) followed by overnight incubation at 4° C rotating. We washed the complexes bound on the beads using buffers with increasing salt concentration: once with “wash A” (20 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA, 1% w/v Triton, 0.1% w/v SDS, once with “wash B” (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% w/v Triton, 0.1% w/v SDS), once with “wash C” (10 mM Tris-HCl pH8.0, 250 mM LiCl, 1 mM EDTA, 1% w/v Nonidet P-40, 1% w/v deoxycholic acid) and twice with TE, followed by treatment with RNAse, proteinase K, reverse the cross-links and precipitation of DNA using ethanol and glycogen.

For Jmjd3 ChIPs, the cells were fixed with 1% formaldehyde for 10 min at 25 °C and lysed on ice using 1ml “cell lysis” buffer (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100)/107 cells. We resuspended the pellet in 1ml buffer II (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA pH 8 and 0.5 mM EGTA)/107 cells. We further resuspended the nuclei in buffer III (10mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% n-lauroylsarcosine) and sonicated with bioruptor (Diagenode) for 40’. Triton was added at a final concentration 1% and the chromatin preparation was precleared using magnetic beads. The antibody (5 mg) was coupled to the magnetic beads (50mls) as in the case of histone marks, the complex was added to the precleared chromatin (the equivalent of 10 million cells/reaction) and the reaction mix was incubated for 12–16 hours. The beads having the immunoprecipitated chromatin fragments were washed 8 times using “RIPA” buffer (50 mM Hepes-KOH pH 7.6, 300 mM LiCl, 1mM EDTA, 1% NP-40 (IGEPAL), 0.7% Na-Deoxycholate) and once with TE. The DNA was cleaned as in the case of the chromatin marks (see above). Libraries were generated as described before36, including end-repair, A-tailing, adapter (Illumina Truseq system) ligation and PCR amplification of the libraries. Ampure XP beads (Beckman Coulter, A63880) were used for DNA cleaning in each step of the process.

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Manufacturer protocol

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