GSM5655993: e12.5_mouse_distal_forelimb_EEDc/-_CBX2_ChIPseq; Mus musculus; ChIP-Seq
ChIP experiments were performed in dissected proximal and distal forelimb buds of CD1 (wild type) and Prrx1Cre; EedDel/Flox (Eedc/-) mice at E12.5 following the same conditions as previously described (Berlivet et al., 2013). Chromatin was cross-linked for 13 minutes using formaldehyde and sonicated using Fisher Scientific, Model 100 sonic dismembrator to obtain fragments between 100-600 bp. Protein A and Protein G Dynabeads (Invitrogen) were incubated for 6 hours at 4℃ with 5ug Suz12 (D39F6, Cell Signaling), 4ug H3K27Ac (ab-4729, Abcam), and 4ug Ring1b (39663, Active Motif). The chromatin was coupled to the beads overnight at 4℃. The immunoprecipitated samples were then washed with different wash buffers depending on the antibody present. For SUZ12 antibody, 6 sequential RIPA washes (50mM Hepes-KOH (pH7.5), 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) were performed. For both H3K27Ac and RING1B sequential washes in low salt (1% Triton, 0,1% SDS, 150 mM NaCl, 20 mM Tris (pH8), 2 mM EDTA), high salt (1% Triton, 0,1% SDS, 500 mM NaCl, 20 mM Tris pH8, 2 mM EDTA), LiCl (1% NP-40, 250 mM LiCl, 10 mM Tris (pH8), 1 mM EDTA) and TE buffer (50 mM NaCl, 10 mM Tris (pH8), 1 mM EDTA) were performed. Lastly, the DNA was purified on QIAquick columns (Qiagen). Library and flow cells were prepared by the IRCM Molecular Biology Core Facility according to Illumina's recommendations and sequenced on Illumina Hiseq 2500 in a 50 cycles paired-end configuration.