GSM5079569: ChIP_wing_Sd_rep2; Drosophila melanogaster; ChIP-Seq
ChIP-seq was performed as previously described (Estella, Mckay, and Mann, 2008) with minor modifications.  Briefly third instar larval heads were dissected and inverted in PBS on ice. Heads were fixed for 20 minutes in 1.8% PFA in crosslinking medium (10 mM HEPES, pH=8.0; 100 mM NaCl; 1 mM EDTA, pH=8.0; 0.5 mM EGTA, pH=8.0) at room-temperature with rotation, and subsequently quenched (Quench solution: 1xPBS; 125 mM glycine; 0.1% Triton X-100). Fixed-heads were then washed 2X in buffer A (10 mM HEPES, pH=8.0; 10 mM EDTA, pH=8.0; 0.5 mM EGTA, pH=8.0, 0.25 % Triton X-100) and 2X buffer B (10 mM HEPES, pH=8.0; 200 mM NaCl; 1 mM EDTA, pH=8.0; 0.5 mM EGTA, pH=8.0; 0.01% Triton X-100) 10 minutes each at 4°C. Wing or haltere discs were then dissected and placed in sonication buffer (10 mM HEPES, pH = 8.0 ;1 mM EDTA, pH = 8.0; 0.5 mM EGTA, pH = 8.0, 0.1 % SDS). Chromatin sonication was performed using a Covaris S2 instrument at settings (105W; 2 % Duty; 15 minutes).  Sonicated chromatin was brought to 1X mild-RIPA (10 mM Tris-HCl, pH=8.0; 1 mM EDTA, pH=8.0; 150 mM NaCl; 1% Triton X-100) concentration and pre-cleared with Dynabeads for 1 hour at 4˚C with rotation. Pre-clearing beads were removed and antibody was added for incubation overnight. Dynabeads were added and incubated for 3 hrs. Bead bound antibody-chromatin complexes were washed as follows 2X RIPA LS (10 mM Tris-HCl, pH=8.0; 1 mM EDTA, pH=8.0; 150 mM NaCl; 1% Triton X-100; 0.1 % SDS; 0.1 % DOC), 2X RIPA HS (10 mM Tris-HCl, pH=8.0; 1 mM EDTA, pH=8.0; 500 mM NaCl; 1% Triton X-100; 0.1 % SDS; 0.1 % DOC), 1X LiCl (10mM Tris-HCl, pH=8.0; 1 mM EDTA, pH=8.0; 250 mM LiCl; 0.5 % IGEPAL CA-630; 0.5 % DOC), 1X TE (10 mM Tris-HCl, pH=8.0; 1 mM EDTA, pH=8.0). Samples were then treated with RNAse and proteinase K, and chromatin was isolated using phenol-chloroform. Imaginal discs of 3rd instar larvae were dissected in PBS on ice.
GEO
GSM5079569