Sc 81599

The ChIP assay was conducted as previously reported [16 (link)]. In short, the cultured microglial cells were washed twice with precooled PBS and added with 1% paraformaldehyde for 5 min of crosslinking at room temperature, which was halted with 127 mM glycine. The cells were then lysed in the ChIP-grade cell lysis buffer containing 50 mM Tris HCl (pH 8.1), 1% SDS, 10 mM ethylenediaminetetraacetic acid (EDTA), and complete protease inhibitor mixture (Roche Diagnostics GmbH, Mannheim, Germany) on ice for 30 min. Then, the cells were subjected to ultrasonic treatment to make the DNA fragments (200–1000 bp). The subsequent ChIP assay was conducted using the ChIP Assay Kit (Millipore). Briefly, 1 μg DNA was added with protein A agarose and ChIP-grade anti-ac-H4 (ab15823, Abcam) or anti-HDAC2 (sc-81599, Santa Cruz Biotechnology) for incubation overnight 4 °C. Normal mouse antibody to IgG (sc-2025, Santa Cruz Biotechnology) or normal rabbit antibody to IgG (sc-2027, Santa Cruz Biotechnology) served as NC. The next day, the DNA complex was washed with low-salt and high-salt buffer and treated with protease K at 56 °C for 2 h, after which the DNA was extracted and purified by phenol/chloroform. The primers for specific ChIP-qPCR amplification of miR-183 promoter region were F: 5′-CGTAGGGCCACTGGACGA-3’ and R: 5′-TTGTCCCCATTCCAGCCCTG-3′.

Cells were crosslinked using 1% formaldehyde and then lysed and subsequently underwent sonication to shear DNA. The ChIP assay was conducted by using a ChIP Assay Kit (Millipore, USA). Also, the antibodies were prepared as follows: anti-HDAC10 (1:100, sc-81599) from Santa Cruz (Santa Cruz, USA), and the negative control was normal IgG (ab172730) from Abcam.