GSM7139590: TTX24h-Smad3; Mus musculus; OTHER
Neurons were dissociated with trypsin. 10,000 cells were counted, centrifuged, and collected for the experiment. Cells were added into ConA beads suspension and incubated for 10 min. The supernatant was discarded. Each sample was added with Antibody Buffer and the corresponding concentration of antibody overnight. After that, supernatant was removed, and each sample was added with a secondary antibody solution and incubated for 1h. The supernatant was separated and pA-Tn5 was added. DNA fragmentation was induced by adding Tagmentation Buffer for 1h. To stop tagmentation and solubilize DNA fragments, 0.5M EDTA, 10% SDS, and 20 mg/mL Proteinase K were added to each sample. DNA fragments were extracted with Tris saturated phenol and chloroform. CUT&Tag was performed with Hyperactive In-Situ ChIP (Vazyme) according to the manufacturer's instructions