GSM3109245: Leg.Dll.Input; Drosophila melanogaster; ChIP-Seq
Third instar larvae were dissected in PBS and fixed for 20 minutes at room temperature in 1.8% formaldehyde, 50mM HEPES, 1mM EDTA, 0.5mM EGTA, 100mM NaCl. After quenching (0.125M Glycine, 1xPBS, 0.01% Triton) the fixed tissue was washed first in Buffer A (10mM HEPES, 10mM EDTA, 0.5mM EGTA, 0.25% Triton, 1mM PMSF), then in Buffer B (10mM HEPES, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.01% Triton, 1mM PMSF), for 10 minutes at 4C.  Imaginal discs were then dissected off the cuticles in Buffer B.  Discs were sonicated in Buffer C (10mM HEPES, 1mM EDTA, 0.5mM EGTA, 1mM PMSF, plus complete protease inhibitor cocktail (Roche)) on ice/EtOH.  Soluble chromatin was transferred to new tubes after centrifugation.  Fresh chromatin, precleared with protein G sepharose beads (Roche), was incubated overnight at 4C with antibody in RIPA buffer (140mM NaCl, 10mM HEPES, 1mM EDTA, 1% Glycerol, 1% Triton X-100, 0.1% DOC, 1mM PMSF).  Antibody-chromatin complexes were pulled down with protein G sepharose beads for 3 hours at 4C.  Bound beads were washed 4-times in RIPA buffer (as above plus 0.1mg/mL ssDNA) and once in TE.  Chromatin was eluted twice in Elution Buffer (1% SDS, 0.1M NaHCO3).  20ug Proteinase K was added, and samples were incubated for 3 hours at 55C, then 65C overnight.  DNA was purified via phenol-chloroform extraction and ethanol precipitation. DNA samples were prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Preparation Kit.
GEO Accession
GSM3109245