GSM1826890: LacO-roX2 rep1; Drosophila melanogaster; ChIP-Seq
For each replicate, 170 male larvae with roX2 inserted into the ectopic 86F8 site were grinded to powder in mortar with liquid nitrogen, and dounced with pestle B in NE buffer (15mM Hepes pH 7.6, 10mM KCl, 5mM MgCL2, 0.1mM EDTA, 0.5mM EGTA, 350mM sucrose, 0.1% Tween, 1mM DTT, protease inhibitors). The nuclei were fixed in 1.8% formaldehyde for 20 min at RT. After quenching for 5 min at RT with 125 mM glycine, nuclei was further dounced for 30 times. The nuclei was then washed 3x 5min in RIPA (25 mM HEPES pH 7.6, 150mM NaCl, 1 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.1% DOC, protease inhibitors). The nuclei was broken with a Branson 250 sonicator at duty cycle 40, intensity 3, and chromatin was sheared by a Covaris S200 sonicator at duty cycle 10, intensity 200 for 30 min. For immunoprecipitation, MOF antibody was added and incubated over night at 4°C. Immunocomplexes were pulled down by protein A-Sepharose. The bead was washed by 4x RIPA buffer, 1x LiCl buffer (10 mM Tris at pH 8, 0.25 M LiCl, 0.5% NP-40, 0.5% DOC, 1 mM EDTA), and 3x TE buffer. The beads were resuspended in TE buffer. The input chromatin and the beads were shaked at 65°C O/N. After 30 min incubation at 37°C with RNaseA (0.2 mg/ml), followed by 2 hr Proteinase K digestion (0.05 mg/ml) at 50°C, DNA was purified using Minelute columns (Qiagen). Pair-end sequencing libraries were prepared using NEBNext® ChIP-Seq library prep reagent kit, according to manufacturer's instruction. Samples were sequenced using Illumina HiSeq 2500.
GEO Accession
GSM1826890