GSM3598064: Ku 120 min control; Drosophila melanogaster; ChIP-Seq
Drosophila embryo extract (DREX) was prepared from preblastoderm embryos (within 90 min after egg laying as described {Becker:1992tt}, with modifications. The embryos were dechorionated in 200 ml embryo wash buffer (0.7% NaCl, 0.04% Triton X-100) and 60 ml 13% sodium hypochlorite (VWR) for 3 min at room temperature (RT) while stirring. Embryos were rinsed for 5 min with cold water and transferred into a glass cylinder with embryo wash buffer. Settled embryos, were washed first in 0.7% NaCl and then in extract buffer (10 mM HEPES, pH 7.6, 10 mM KCI, 1.5 mM MgCl2, 0.5 mM EGTA, 10% glycerol, 10 mM 3-glycerophosphate; 1 mM DTT, protease inhibitors added freshly before use (0.2 mM PMSF, 1 mM Aprotinin, 1 mM Leupeptin, 1 mM Pepstatin). Embryos were settled in a homgenplus homogenizer (Schuett-Biotec) the supernatant was decanted and homogenized with one stroke at 3000 rpm and 10 strokes at 1500 rpm. The homogenate was filled up to 5 mM MgCl2 (f.c.) and centrifuged for 15 min at 27,000 g at 4°C. The white lipid layer was discarded and the supernatant was centrifuged for 2 h at 245,000 g at 4°C. The clear extract was collected with a syringe, leaving the lipid layer and pellet behind. MNase-cleaved DNA after overnight proteinase K digestion was purified with the GenElute PCR clean-up kit (Sigma-Aldrich) and libraries for paired-end sequencing were prepared using the MicroPlex Library Preparation kit (Diagenode). Fifty bp sequencing reads were obained on a HiSeq 1500 (Illumina) instrument.
GEO Accession
GSM3598064