ChIP-seq of E2f, Max, Myb and Myc in Drosophila melanogaster S2 cells for mapping their genome-wide binding sites
ChIP-seq of E2f, Max, Myb and Myc in Drosophila melanogaster S2 cells for mapping their genome-wide binding sites
Chromatin immunoprecipitation (ChIP)-assays were done as in Tuupanen et al.Nature Genetics 41,  885, 2009. ChIP-seq was performed essentially as described in (Tuupanen et al.Nature Genetics 41,  885, 2009) with the following modifications. ChIP for Myc and Max: S2 cells were formaldehyde crosslinked, sonicated and immunoprecipitated with the following set of antibodies (Santa Cruz Biotechnology, Inc.): Myc: dN-20 and d1-717, sc-15832 and sc-28207. Max: d1-160, sc-28209. IgG controls sc-2027 and sc-2028. ChIP for E2f and Myb: S2 cells were transiently transfected with plasmids (backbone pRmHa-1) containing metallothionein promoter driven Cterminally His-streptavidin binding peptide-3xV5 tagged Drosophila open reading frames. Expression of the genes was induced 24 hr after transfection by 0.5 mM CuSO4, and the incubation continued for 48 hr until formaldehyde crosslinking was done. Immunoprecipitations were made with 7.5 mg of monoclonal anti-V5 antibody (46-1157; Invitrogen) or IgG control antibody (sc-2025; Santa Cruz Biotechnology, Inc.) Chromatin immunoprecipitation (ChIP)-seq libraries were prepaired as in Tuupanen et al.Nature Genetics 41,  885, 2009.  Library preparations were done by repairing the immunoprecipitated DNA using Klenow and T4 DNA polymerase and T4 polynucleotide kinase (MBI Fermentas, Vilnius, Latvia), and ligating the Illumina sequencing adapters according to manufacturers instructions.