Fine Mapping of Genomic Targets

The following protocol has been established for the cultured cell line Schneider 2 (S2) from Drosophila melanogaster. If other tissue is used, certain steps of the protocol may need to be optimized, although the basic rationale would be the same. The main difference is likely to be in the cross-linking step. Cells growing in suspension can be easily cross-linked by adding a concentrated stock of the cross-linking buffer directly to the growth medium (see below). The same procedure is used for yeast (Strahl-Bohlsinger et al. 1997). Adherent mammalian cells are also cross-linked by adding a concentrated stock of the cross-linking buffer to the medium. After cross-linking and quenching with glycine, the cells are scraped and washed off the culture dishes with PBS and pooled. Then cells are lysed according to the protocol below (see Fig. 2). When using more compact material, such as embryos or imaginal discs, cross-linking conditions are more vigorous and might include treatments with detergents or polar solvents, which allow the formaldehyde to better penetrate the sample. For additional protocols, see Cao et al. (2002; imaginal discs from D. melanogaster), Orlando et al. (1998; embryos from D. melanogaster), and Chua et al. (2004; tobacco shoots). For further specialized protocols, see also the Web sites listed after the reference section.