title: GSM7519048: POLR1A_2cell_early_rep3; Mus musculus; ChIP-Seq
library_construction_protocol: A hyperactive in-situ ChIP assay was performed in accordance with manufacturer's instructions (Vazyme, TD901-01). Briefly, ConA-beads were washed once with binding buffer and incubated with zona pellucida-removed 2-cell embryos. Bead-bound cells were resuspended in antibody buffer containing a primary rabbit monoclonal anti-POLR1 antibody (24799, Cell Signaling Technology) or negative control IgG (sc-2025, Santa Cruz) for 2 h with slow rotation. The primary antibody was removed using a magnet stand. Then, bead-bound cells were incubated in Dig-wash buffer containing a secondary rabbit anti-mouse IgG H&L antibody (ab6709, abcam). Cells were incubated with Hyperactive pA-Tn5 Transposase adapter mix and then resuspended in tagmentation buffer. DNA was purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation, and treated with RNase A. Then, library amplification and PCR product purification were performed. A hyperactive in-situ ChIP assay was performed in accordance with manufacturer's instructions (Vazyme, TD901-01). Briefly, ConA-beads were washed once with binding buffer and incubated with zona pellucida-removed 2-cell embryos. Bead-bound cells were resuspended in antibody buffer containing a primary rabbit monoclonal anti-POLR1 antibody (24799, Cell Signaling Technology) or negative control IgG (sc-2025, Santa Cruz) for 2 h with slow rotation. The primary antibody was removed using a magnet stand. Then, bead-bound cells were incubated in Dig-wash buffer containing a secondary rabbit anti-mouse IgG H&L antibody (ab6709, abcam). Cells were incubated with Hyperactive pA-Tn5 Transposase adapter mix and then resuspended in tagmentation buffer. DNA was purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation, and treated with RNase A. Then, library amplification and PCR product purification were performed.