GSM9108692: Sample_WJ-3830-Ras17-6-Input, rep 1; Drosophila melanogaster; ChIP-Seq
Cells were crosslinked by adding the formaldehyde solution directly to the cell culture to a final concentration of 1.8% and incubating for 10 minutes at 25C. The reaction was stopped by adding glycine pH 7.0 to a final concentration of 0.125 M. Cells were extensively washed and 0.5 ml of pelleted crosslinked cells were resuspended in 5 ml cold TE buffer. Cell suspension was supplemented with 0.1u/ml MNase and 2mM CaCl2 and incubated at 37C for 25 min. The treated cells were lysed by ultrasound and resulting lysates adjusted to RIPA (140mM NaCl, 10mM Tris-HCl pH8.0, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% DOC) ChIP was done essentially as described in (Schwartz et al., 2006) with the following modifications. Crosslinked cell lysates containing 100µg of DNA was used for immunoprecipitations in a total volume of 500µl of RIPA. The resulting immunoprecipitated DNA was dissolved in 40µl of water for ChIP-seq library preparation. 2ng of immunoprecipitated DNA were processed using NEBNext Ultra II DNA Library preparation Kit for Illumina (cat# E76455) and index oligonucleotides from NEBNext Multiplex Oligos for Illumina (cat# E7335).