Fig. 1

A scheme explaining the difference between the xChIP-seq and xxChIP-seq protocols. Note that the DNA sequences that are associated with “hidden” epitopes in situ are revealed by xChIP-seq, but not with xxChIP-seq. This scheme does not specify the type of chromatin higher-order structure involved in creating the “hidden” H1 epitope. “Masking” proteins, capable of “blocking” the H1 epitope in vivo, certainly can exist. Probably, the first formaldehyde fixation will covalently attach these proteins to H1 (and mask the epitope), such that the sonicated products cannot be immunoprecipitated at any point in the protocol. The xxChIP-seq versus the xChIP-seq comparison depends upon the differences in “exposed” H1 epitopes. If the masking protein is “knocked-off” during the xChIP sonication, exposing the H1 epitope, it will “mimic” higher-order structure, which is (presumably) also destroyed during the xChIP sonication, exposing H1 epitopes. “Higher-order chromatin structure” is only detected after anti-H1 binding, washing, a second formaldehyde fixation and sonication (xxChIP). Any exposure of H1 epitopes, at this point, will be undetected, since there is no further incubation with anti-H1 antibodies. Immunoprecipitation occurs because the Protein A/G agarose captures chromatin fragments by their covalently bound IgG (anti-H1) molecules