miR-1306 Mediates the Feedback Regulation of the TGF-ß/SMAD Signaling Pathway in Granulosa Cells

Yang, Liu; Du, Xing; Liu, Lu; Cao, Qiuyu; Pan, Zengxiang; Li, Qifa

In total, 1 × 10 7 porcine GCs were collected and 1% formaldehyde was added to crosslink the protein and the chromatin. The collected cells were placed on ice and sonicated for 5 min (10 s interval on and off) by 30% output control with a 3-mm microtip. The sonicated protein-chromatin complex (~800 µL) was centrifuged at 10,000 rpm and 100 µL of the supernatant were collected as input control. Then, 400 µL of the supernatant were diluted 2.5-fold and incubated with 100 µL protein A/G-agarose (Santa Cruz, #sc-2003) by shaking for an hour at 4 • C. This mixture was then centrifuged and the purified supernatant was transferred into a new dolphin tube. Then, 10 µL (~4 µg) of anti-SMAD4 antibody or rabbit IgG antibody were added into each sample and incubated by shaking at 4 • C overnight. The reversal of crosslinking was done using 16 µL NaCl (5M) at 100 • C for 10 min and the proteins were dissolved with 10 µL proteinase K at 50 • C for an hour. Then, DNA was released and precipitated with chloroform-isoamylalcohol method. Following, the enrichment of miR-1306 promoter was determined by using qRT-PCR. The primers for chromatin immunoprecipitation (ChIP) are listed in Supplementary Table S4 .