This protocol describes a tandem affinity chromatin immunopurification strategy to analyze the distribution of chromatin remodelers onto the mouse genome. It is based on mouse embryonic stem (ES) cell lines that express, from the endogenous loci, remodelers fused with a tandem affinity purification (TAP)-tag. Two versions of the TAP-tag, introduced at the C terminus, have been used successfully: (FLAG3-TEV-HA) and (FLAG-HA).
ES cells are first fixed either with formaldehyde, or with a combination of disuccinimidyl glutarate (DSG) and formaldehyde. ES cells are then permeabilized, and incubated with micrococcal nuclease (MNase) to fragment the genome into mononucleosomes. These nucleosomes are then incubated with agarose beads coupled with an antibody against HA or FLAG epitopes. After a series of washes, tagged-remodeler-nucleosome complexes are eluted, either by TEV protease cleavage or by peptide competition. The eluted protein is then subjected to a second immunopurification step, using beads coupled to the antibody against the second HA or FLAG epitope. After elution, DNA is extracted from the highly purified mononucleosome fraction, and processed for high-throughput sequencing.