Chip kit

ChIP was conducted by using a ChIP kit (Qiagen, Düsseldorf, Germany) in accordance with the manufacturer’s instructions. In brief, the cells were incubated with formaldehyde, and the link between DNA and protein was built. Then, the cross-linked chromatin was sonicated into fragments. Anti-H3K27ac antibodies (Abcam) were used for the immunoprecipitation of the chromatin fragments, and IgG was used as the negative control. Subsequently, the precipitated chromatin DNA was measured through qRT-PCR.

Preadipocytes isolated from inguinal fat were synchronized to G₁ phase as described the above. After the cells were re-fed with 10% calf serum for 8 h, cells were fixed with formaldehyde (final concentration 1%) for 10 min. Fixation was then stopped with the addition of glycine. The remainder of the procedure followed the manufacturer’s instructions for ChIP Kit (Qiagen Inc). For each ChIP reaction, 2 million cells were used. The antibodies used were anti-THRA (ab2743 ChIP grade, rabbit, Abcam Inc.), anti-NCoR (17-10260, ChIP grade, rabbit, Millipore Inc.), and anti-CREB (ab31387, ChIP grade, rabbit, Abcam Inc.). Normal rabbit IgG was used as negative control. The Input was 10% pre-cleared total lysate. The purified DNA was PCR amplified for 35 cycles with triplicates. The data shown was deducted from negative control and expressed as % Input enrichment. ChIP q-PCR primers were Qiagen pre-designed and are shown (Table 1).