Histones were identically tagged at their N-termini with biotin ligase recognition peptide (BLRP) (de Boer, E. et al. Proc Natl Acad Sci U S A 100, 7480-5 (2003), and Beckett, D. et al. Protein Sci 8, 921-9 (1999)) and expressed together with E. coli biotin ligase in Drosophila S2 cells. Nuclei were prepared as described (McKittrick, E., et al., Proc. Natl. Acad. Sci. USA 101, 1525-1530 (2004).), and treated with micrococcal nuclease (MNase) to digest chromatin to yield mostly mononucleosomes. Chromatin was extracted (input) using a modification of the procedure of Blower, M.D., et al. (Developmental Cell 2, 319-330 (2002)). Biotinylated histone-containing nucleosome particles (pull-down) were purified and DNA was extracted from both input and pull-down fraction for microarray analysis. DNA from input (3 ug) and pull-down (4 ug) was provided to NimbleGen Systems for differential labelling by random priming with Cy3- or Cy5 and hybridization to oligonucleotide arrays. For each experiment, input and pull-down channel signal intensities and scaled log2(ratios) were provided by NimbleGen Systems.
Data processing
Medians of Cy5 and Cy3 signals and backgrounds were computed, the median background was subtracted from the median signal, and the log base 2 of the ratio was computed
