GSM1313421: ChIP-seq of ZIPIC in S2 cells; Drosophila melanogaster; ChIP-Seq
Samples of 107 S2 cells in 10 mL of SFX medium were treated with 37% formaldehyde added to a final concentration of 1% and incubated on a rotator at room temperature for 10 min. Cross-linking was stopped by 0.125 M glycine, and the samples were washed with three portions of PBS (pH 8), with 0.5 mM PMSF and pelleted at 1000 rpm, 4°C, for 5 min. The pellet was resuspended in 10 mL of buffer I (25 mM HEPES, pH 7.8; 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF, Calbiochem Cocktail V) and placed on ice for 10 min. The suspension was then homogenized by 20 strokes in Dounce homogenizer with pestle B and centrifuged at 2000 rpm, 4°C, for 5 min. The pellet was resuspended in 5 mL of buffer II (50 mM HEPES, pH 7.8; 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5 mM PMSF, Calbiochem Cocktail V), sonicated in a Bioruptor sonifier (Diagenode, Belgium) for 15 min, with alternating 30-s ON/OFF intervals, and centrifuged at 14 000 rpm, 4°C, for 5 min to remove cell debris. A 50-µL aliquot of the supernatant was used to test the results of sonication and measure DNA concentration: the sample was diluted with 350 µL of elution buffer  (50 mM Tris, pH 8.0; 1 mM EDTA, 1% SDS, 50 mM NaHCO3) and treated with RNase A (1 µL from 10 mg/mL stock) at 37°C for 1 h and then with proteinase K (1 µL from 20 mg/mL stock) at 42°C for 2 h. After subsequent incubation at 65°C for 6 h, DNA was isolated by phenol–chloroform–isoamyl alcohol extraction, concentrated by ethanol precipitation with glycogen (each tube was supplemented with 5 µL of glycogen (from 20 mg/mL stock), 40 µL of sodium acetate (from 3 M stock), and 1 mL of ethanol, vortexed, and placed at –20°C for 4–5 h). The precipitated DNA was pelleted at 14 000 rpm for 30 min, washed with 80% ethanol, and resuspended in 50 µL of MilliQ water. Protein A Sepharose (Pierce) was washed with three portions of buffer II and incubated with 1 mg/mL BSA in the same buffer on a rotator at 4°C for 4 h. Chromatin samples containing 10–20 µg of DNA equivalent were each diluted with buffer II to a final volume of 1 mL, their 50-µL aliquots were stored as input, and then the samples were incubated overnight, at 4°C, with appropriate antibodies (rabbit antibodies against Pita (1:1000), ZIPIC (1:200), or CP190 (1:500), or with nonspecific IgG purified from rabbit preimmune sera (control)) in the presence of blocked Protein A Sepharose beads (40 µL). After incubation, the beads were washed on a rotator, at 4°C, with three 1-mL portions of buffer II and two 1-mL portions of buffer III (50 mM HEPES, pH 7.8; 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1 % SDS, 0.5 mM PMSF, Calbiochem Cocktail V), each wash for 10 min. Then 400 µL of elution buffer was added, and the beads were treated with RNase A (1 µL from 10 mg/mL stock) at 37°C for 1 h and, after adding 20 µL of NaCl (from 4 M stock), with proteinase K (1 µL from 20 mg/mL stock) at 42°C for 2 h. The samples were incubated on a thermoshaker at 65°C overnight, and DNA was isolated by phenol–chloroform–isoamyl alcohol and chloroform–isoamyl alcohol extraction in phase-lock tubes. The samples were concentrated by ethanol precipitation with glycogen (as described above), and resuspended in MilliQ water. Sequencing libraries were prepared from 10 ng of immunoprecipitated DNA with the Illumina ChIPSeq DNA Sample Prep Kit according to Illuminas' instructions
GEO Accession
GSM1313421