title: GSM2060319: CS2_H3K4me3 [ChIP-seq]; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Homogenateswere placed in Bio-Spin columns (Bio-Rad) and centrifuged at 1000g for 4 min. Filtered homogenates were then centrifuged at 6000g for 10 min. The nuclei-containing pellets were resuspended in 1 mL of NEB and centrifuged at 20,000g for 20 min on sucrose gradients (0.65 mL of 1.6 M sucrose in NEB, 0.35 mL of 0.8 M sucrose in NEB). The pellet was resuspended in 1 mL of NEB and 11% formaldehyde (diluted in Schneider's media; Sigma) was added to a final concentration of 1%. Nuclei were cross-linked for 10 min at room temperature and quenched by adding 1/10 volume of 1.375 M glycine. The nuclei were collected by centrifugation at 6000g for 5 min. Nuclei were washed twice in 1 mL of NEB and resuspended in 450 μL of Sonication buffer (10 mM HEPES, pH 7.5, 2 mM EDTA, 1% SDS, 0.2% Triton X-100, 0.5 mM spermidine, 0.15 mM spermine) and sheared by sonication three times for 7 min using a Bioruptorsonicator (Diagenode) on ice. Sonicated samples were centrifuged at 15,000g for 10 min and frozen at −80°C in 150-μL aliquots. Of the sonicated chromatin, 25 µL was used as input control. The remaining 125 μL were diluted in 1.1 mL of IP buffer (50 mM HEPES/KOH, pH 7.6, 2 mM EDTA, 1% Triton, 0.1% deoxycholate). Antibody was added: 4 µg of anti-H3K4met3 antibody (abcam8580), and samples were rotated at 4 °C overnight Protein A/G-plus beads (Santa Cruz biotechnology) were blocked overnight in 0.1 mg/mL yeast tRNA and 1 mg/mL BSA in IP buffer. After overnight incubation, the beads were washed once in IP buffer, added to the chromatin/antibody mixture, and then incubated for an additional 2 h at 4°C.Beads were centrifuged at 1,000 rpm for 20s and were washed once in 1.5 mL of ChIP Wash buffer (50 mM HEPES-KOH, pH 7.6, 1 mM EDTA, 1% Triton, 0.1% deoxycholate, 0.1% Sarkosyl, 0.1% BSA, 0.5 M KCl). Beads were resuspended in 1 mL of ChIP Wash buffer and rotated for 30 min at 4°C. Beads were then washed once in Li Wash Buffer (10 mM Tris, pH 8.0, 0.25 M LiCl, 0.5% NP40, 0.5% deooxychoalte, 1 mM EDTA) and once in cold TE (pH 8.0) before being eluted with 150 μL of ChIP Elution buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1% SDS, 1 mM DTT, 0.1 mg/mL proteinase K). ChIP Elution buffer (150 μL) was also added to the input sample. Both IP and input samples were incubated for 2h at 37°C. The sepharose beads were removed from the IP samples, and cross-links were reversed by addition of 20µL 5M NaCl and incubation overnight at 65°C. DNA was isolated from the samples using phenol-chloroform (Sigma) extraction. DNA library preparation was performed as described by Blecher-Gonen et al. : "High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states".
attributes.tag: GEO Accession
attributes.value: GSM2060319