The DNA levels were normalized to those of the appropriate chromatin inputs after they are subtracted with DNA signals given by ChIP using normal rabbit or goat IgG.
For the negative control, we used normal rabbit IgG (Santa Cruz Biotechnology) instead of the antibody.
Nonspecific binding was blocked by using 10% normal rabbit serum and 0.5%.
IEM controls using normal mouse or rabbit serum, or antibodies against irrelevant proteins, were all negative.
Control ChIP was performed using a normal rabbit IgG (Santa Cruz, Santa Cruz, CA).
Serum blocking was performed using 10% normal rabbit serum for 30 min at room temperature.
Anti-ERα antibody (Epitomics) was used and normal rabbit IgG (Sigma) was used as negative control.
In addition, for negative controls, we used normal mouse sections subjected to normal rabbit serum as the primary antibody.
For protein measurement, MRK16 anti-Pgp (Kamiya Biomedical) antibody (30 minutes, room temperature) was used followed by 20% normal rabbit serum to block (30 minutes, 4°C) and FITC-conjugated goat anti-mouse secondary antibody (30 minutes, 4°C; Dako).
ChIP with anti-Twist1 was performed in E12.5 ECCs, E10.5 limb buds, and PNST cells followed by quantification using qPCR relative to normal rabbit IgG control.
