Genome-wide maps of GlcNAc bound proteins and Pho ChIP-seq in Drosophila S2 cells and pupae.. Drosophila melanogaster

We developed a strategy to map GlcNAc modified proteins based on a chemical chromatin precipitation strategy. Drosophila S2 cells, as well as wild type (wt) and sxc null pupae, were fed with 100 uM Ac4GalNAz and the resulting DNA was cross-linked, sonicated, and purified. DNA strands cross-linked to GlcNAz proteins were isolated by congugating with alkyn-biotin by Staudinger ligation followed by streptavidin purification. The resulting DNA was constructed into libraries for sequencing. To asses the robustness of our strategy, we compared GalNAz ChIP-seq results in S2 cells with two other GlcNAc ChIP-seq strategies, using a mutant β-1,4-galactosyltransferase (GalT) and the lecting wheat germ agglutinin (WGA). Briefly, GalT was incubated with cross-linked, sonicated and purified DNA along with UDP-GalNAz. Ligation to biotin with click chemistry followed by streptavidin purification resulted in library ready material. For WGA and Pho ChIP-seq, cross-linked and purified DNA was incubated with pho antibody or WGA resin and purified followed by library preperation. Overall design: Examination GlcNAc bound loci in Drosophila cells and pupae.