Normal mouse anti igg

AC-16 cells were lysed using RIP buffer, and then incubated with RIPA buffer containing magnetic beads conjugated with human Anti-Ago2 antibody (Millipore, Billerica, MA, USA) or normal mouse Anti-IgG (Millipore). After interaction with Proteinase K, the immunoprecipitated RNA was extracted and purified RNA was determined using qRT-PCR. All experiments were repeated three times independently.

As specifical immunoprecipitate CP2 antibodies are not available, we constructed pCMV-C-HA-CP2-CDS and then transfected this vector into mGCs. ChIP was performed using the EZ-ChIP Kit (Millipore). The AVCX130 system (Sonics & Materials, Newtown, CT, USA) was used for cell sonication. Anti-HA (Abcam, ab9110), anti-OCT1 (Santa Cruz Biotechnology, sc-25399 X) and normal anti-mouse-IgG (Millipore) were used for the immunoprecipitation reactions. DNA from the immunoprecipitated complex was amplified via PCR. The primer sequences are described in Supplementary Table S2.

ChIP was performed using the Magna ChIP A/C kit (Millipore, catalog no. 17-10086) according to the manufacturer’s instructions. In brief, 1 × 10⁷ DF-1 cells of 10-cm dishes were transfected with pCMV-C-HA-AhR:Arnt or pCMV-C-HA for 36 h, and then fixed with 1% formaldehyde for 10 min at room temperature. Subsequently, the lysates were sonicated and then immunoprecipitated with normal anti-mouse-IgG (Millipore, St. Louis, MO, USA) as a negative control or with the antibody against HA (Abcam, Cambridge, MA, USA, ab9110); 2 μg of antibody per 25 μg of DNA was used. The captured chromatin was eluted and un-cross-linked by heating at 62 °C, and then the immunoisolated DNA was subjected to qPCR amplification using primers (Table 1), TransStart Top Green qPCR SuperMix (TRANSGEN, China), and a real-time PCR detection system (Bio-Rad). The input DNA used for qPCR was equivalent to 0.01% of the original sample used for the ChIP assay.

For ChIP assay, the EZ-Magna ChIP A/G kit (Millipore) was used following the instructions as previously described by the manufacturer. The anti-EZH2 (Active Motif) and anti-H3K27me3 (Active Motif) were applied. Anti-normal mouse IgG (Millipore) and anti-RNA polymerase-II (Abcam) were used as negative control, or positive control, respectively. Primers for ChIP-qPCR are listed in Supplementary Table 3.