Drosophila S2 and S2R+ cells were grown to log phase in Schneider’s media (Gibco) supplemented with 10% FBS (HyClone). Drosophila DmBG3-c2 (BG3) cells were grown in M3 media (Sigma) supplemented with BPYE, 10% FBS, and 10 µg/mL insulin. Nuclei were harvested and digested with MNase as previously described (Henikoff et al. 2009 (link)). MNase-digested chromatin was then extracted and solubilized in 80 mM NaCl buffer by pushing through a 26-gauge needle as described (Kasinathan et al. 2014 (link)). Antibodies against Polycomb (Schuettengruber et al. 2009 (link)), TRL (Melnikova et al. 2004 (link)), ADF1 (Lang and Juan 2010 (link)), PHO (Klymenko et al. 2006 (link)), and H3K27me3 (Abcam, ab6002) were used for immunoprecipitation. Illumina sequencing libraries, paired-end sequencing, and base calling were performed as described using TruSeq primers (Henikoff et al. 2011 (link)).
Protocol detail
Drosophila Chromatin Immunoprecipitation and Sequencing
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Antibodies
Buffer
Cells
Chromatin
Drosophila
Immunoprecipitation
Insulin
Nacl
Needle
Nuclei
Polycomb
Primers
Corresponding Organization :
Other organizations : Institut Curie, Centre National de la Recherche Scientifique, Harvard University, University of Washington, Fred Hutch Cancer Center, Duke University, Duke University Hospital, Duke Medical Center, Howard Hughes Medical Institute
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Variable analysis
independent variables
- Cell type (Drosophila S2, S2R+, and BG3)
- Cell culture media (Schneider's media and M3 media)
- Chromatin digestion by MNase
- Chromatin extraction and solubilization
- Immunoprecipitation of protein targets (Polycomb, TRL, ADF1, PHO, and H3K27me3)
- Cell culture conditions (growth to log phase)
- Cell culture supplements (10% FBS, BPYE, and 10 µg/mL insulin)
- Extraction and solubilization method (pushing through 26-gauge needle)
- Positive control: Experimental procedures described in previous publications (Henikoff et al. 2009, Kasinathan et al. 2014)
- Negative control: Not explicitly mentioned
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