GSM2536239: CTCF ChIP-seq (control); Drosophila melanogaster; ChIP-Seq
S2 wildtyp cells were seaded 2x 10^7 cells/ 20 ml flask. Inducible cells were induced with 100mM CuSO4 24 h after seading cells. After 48 h they were harvested and counted. The ChIP was performed as described previously (Bartkuhn et al. 2009) with minor modifications in sonication. The cells were fixed by 1 % formaldehyde. Fixation was stopped by the use of 0,125M Glycin. After washing cells with 1x PBS, lysis buffer (1% SDS, 10mM EDTA, 50mM Tris/HCl pH 8, supplemented with Roche proteinase inhibitor cocktail) was added to the cells and the DNA was fragmented with the Bioruptor sonicator (Diagenode). Cells were incubated for 2x 10 cycles (30 sec sonication, 30 sec pause) within the ice cooled sonicater. The resulting fragments ranged from 200 – 500 bp. Chromatin samples were diluted 1:10 by dilution buffer (0,01% SDS, 1,1% Triton X-100, 1,2% EDTA, 16,7mM Tris/HCl pH 8, 167mM NaCl in ddH2O), with addition of glycerol for stability of the chromatin. 10 % input samples were separated from prepared chromatin and were stored at -20°C. Pre-clearing of Chromatin was performed with protein A/G agarose beads (Millipore) for 45 min at 4°C. IP with specific antibodies for CP190, dCTCF, FLAG and Histone H3 was performed at 4°C. Mouse IgG was used as IP control. After 4 h beads were added and the solution was further incubated at 4°C o. n. The beads were than washed by different salt buffers with increasing salt concentration, low salt buffer (0,1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris/HCl pH 8, 150mM NaCl in ddH2O), high salt buffer (0,1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris/HCl pH 8, 500mM NaCl in ddH2O) and LiCl buffer (0,25% LiCl, 1% NP40, 1mM EDTA, 10mM Tris/HCl pH 8, 1% DOC in ddH2O), as well as 2x TE buffer (10mM Tris/HCl pH 8, 1mM EDTA). Clean up of DNA was performed with 100 µl Chelex 100 agarose beads (BioRad) to gain higher DNA amount for sequencing of ChIP-DNA. Proteinase K digestion was done with 10 µg/ml of the enzyme during Chelex DNA extraction. DNA from 10 % Input samples were also extracted by Chelex method (Nelson et al. 2006). Sequencing libraries were prepared with the NEBNext ChIP-Seq Library Prep Reagent (New England Biolabs) according to manufacturer’s instructions.