GSM8068306
Harvesting mouse liver,excising desired size, and flash freezing on liquid nitrogen. Extracting nuclei from the tissue according to manufacturer's instruction. CUT&Tag assay was performed using HyperactiveTM In-Situ ChIP Library Prep Kit for Illumina (cat.no.TD901-TD902, Vazyme Biotech, China) according to manufacturer's instruction. Prepared concanavalin A-coated magnetic beads (ConA beads) were added to resuspended cells and incubated at room temperature to bound cells. Non-ionic detergent Digitonin was used to permeate cell membrane. Primary anti-H3K27ac antibody (ab177178, Abcam), secondary antibody and the Hyperactive pA-Tn5 Transposase were incubated with the cells that were bounded by ConA beads in order. Therefore, the Hyperactive pA-Tn5 Transposase can exactly cut off the DNA fragments that were bound with target protein. In addition, the cut DNA fragments can be ligated with P5 and P7 adaptors by Tn5 transposase and the libraries were amplified by PCR with the P5 and P7 primers. The purified PCR products were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Finally, these libraries were sequenced on the Illumina NovaSeq6000 platform and 150bp paired-end reads were generated for the following analysis.