Hyperactive pg mnase cut run assay kit for pcr qpcr

For RT-qPCR, the total RNA of cells was extracted by Trizol method, followed by cDNA synthesis of 1 μg of RNA using StarScript III All-in-one RT Mix with gDNA Remover Kit (GenStar). SYBR-Green-based qPCR was performed to measure the expression of genes. The mRNA expression level was normalized by the expression level of GAPDH.
For pG-MNase CUT&RUN for PCR/qPCR, follow the manufacturer’s instructions for Hyperactive pG-MNase CUT&RUN Assay Kit for PCR /qPCR (HD101, Vazyme Biotech). In brief, the cells were collected and counted, and incubated with pre-treated ConA Beads Pro. Cells are resuspended in Antibody Buffer and incubated overnight at 4 °C with the primary antibody. The pG-MNase Enzyme was rotated at 4 °C and incubated for 1 h. After fragmentation, DNA was terminated and released. FastPure gDNA Mini Columns were used to extract DNA, and then quantitative detection by qPCR was performed. The expression levels of the genes were normalized with the Spike in levels that had been added.
The sequences of all qPCR primers mentioned in this paper can be obtained in the supplementary data 5.

CUT&RUN-qPCR is a new method to extract the DNA bound to protein. NSCs stably expressing Mysm1-Flag and induced astrocytes were harvested and treated according to the manufacturer’s instructions by using a Hyperactive pG-MNase CUT&RUN Assay Kit for PCR/qPCR (HD101, Vazyme Biotech). In brief, cells were collected to combined with ConA Beads Pro. Subsequently, cells were resuspended in antibody buffer and incubated with primary antibodies against Flag (F1804, Sigma‒Aldrich), ubiquityl-Histone H2A (#05-678, Millipore), Tri-Methyl-Histone H3 (Lys4) (#9751, Cell Signaling Technology), or Acetyl-Histone H3 (Lys9) (#9649, Cell Signaling Technology). Antibody-protein complexes were incubated with pG-MNase Enzyme. Then fragmentation was terminated and released by Stop Buffer containing Spike in DNA. The DNAs were extracted by FastPure gDNA Mini Columns and subjected to qPCR. The Spike in DNA was used as a reference and the primer sequences for CUT&RUN-qPCR are listed in Supplementary Table 3. All cells were tested for mycoplasma contamination before experiment.

The Hyperactive pG‐MNase CUT&RUN Assay Kit for PCR/qPCR (Vazyme, HD101‐01) was used to conduct the CUT&RUN assay. Some 1 × 10⁵ HCC cells were gathered, washed once in 100 µL of washing buffer, then bound to ConA beads for 10 min at 25°C. Cells were then treated with 1 µL of SP1 (CST, #9389) antibody at 4°C overnight. The following day, pG‐MNase enzyme was added, and the cells were incubated for 1 h at 4°C after being washed twice with DIG washing buffer. Following two rounds of DIG washing, cells were incubated with CaCl₂ for 1 h at 4°C. We added 100 mL of stop buffer, resuspended the cells, and incubated the specimens for 30 min at 37°C. Cells were plated on a magnet after incubation, and any unattached liquid was drained. DNA was eluted from the beads with double‐distilled water after they had been gently washed twice with 80% ethanol. qRT‐PCR tests were conducted in accordance with the manufacturer's recommendations.