title: GSM2266308: H2A.C ChIP; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Unstaged embryos (1 g) were dechorinated in 120 ml, 1:5 diluted, sodium hypochloride (VWR, Cat.no. 301696S) for 3 min. The embryos were thoroughly washed and fixed in the fixing solution [10 ml, 0.1 M NaCl, 0.05 M HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 3.7% Formaldehyde (Sigma, Cat. No F1635) added to 30 ml n-Heptane (VWR, Cat. No. 8.22332.1000)] for 15 min at 16-18°C on a rotating wheel. Fixation was quenched by adding 125 mM glycine. The embryos were subsequently washed with PBS (including 0.01% Triton-X100) for 10 min and stored at -80°C until further use.   For nuclei isolation, embryos were slowly thawed and dounced using a glass homogenizer (Schubert, Cat.no. 9164693) with 20 strokes each of the A and B pestles in ice-cold NX-I buffer [15 mM HEPES pH 7.6, 10 mM KCl, 2 mM MgCl2, 0.5 mM EGTA, 0.1 mM EDTA, 350 mM sucrose, 1 mM DTT, 0.2 mM PMSF, Protease inhibitors Leupeptin, Pepstatin and Aprotinin (10 µg/ml)]. The lysate was filtered through Miracloth and nuclei were pelleted at 3500 rpm, 10 min, 4°C. The pellet was washed in NX-I buffer. Finally, the nuclei were resuspended in RIPA [1% Triton X-100, 0.1% Sodium deoxycholate, 140 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.1% SDS, 1 mM PMSF] and washed 3 times. Nuclei were then counted and frozen at -80°C in aliquots of ~109 nuclei/ml.  For shearing and ChIP, thawed nuclei were adjusted to ~2x108/ml by dilution in RIPA and sheared with a Covaris S220 system (Covaris Inc. MA, USA) at 110 Watts, 20% duty factor and 200 cycles per burst for 25 min. Chromatin was pre-cleared using protein a A+G bead (1:1) mix for 1h at 4°C. Immunoprecipitations were set up overnight at 4°C with 200 µl chromatin and 4 µl of the respective antibody adjusted to 500 µl with RIPA. RIPA-equilibrated protein A+G (1:1) mix was then added to precipitate the immune-complexes for 3 h at 4°C. Beads were washed subsequently 5 times for 10 min each in 1 ml RIPA buffer. Residual RNA was digested by RNase A (10 µg/100 µl, Sigma, Cat. No. R4875) at 37°C for 20 min. Subsequent protein digestion (25 µg/100 µl, Proteinase K, Genaxxon, Cat.no. M3036.0100) and reversal of cross-linking were performed together at 68°C for 2 hours. DNA was purified using GenElute™ PCR Clean-Up Kit (Sigma, Cat.no NA1020) ChIP DNA was quantified using the Qubit® dsDNA HS Assay Kit (Life Technologies, Cat.no.Q32851) and sequencing libraries were prepared using the MicroPlex Library Preparation kit (Diagenode, Cat. No. C05010011) starting from 2 ng DNA, whenever possible. PCR amplification was monitored by quantifying amplified libraries (maximum 19 cycles).
attributes.tag: GEO Accession
attributes.value: GSM2266308