ChIP–qPCR was performed using the ChIP-IT High Sensitivity kit (Active Motif #53040) according to the manufacturer’s instructions, except that inputs were purified using a clean and concentrate column (Zymo Research #D5205), and the ChIP samples were eluted twice with 30 μL each for a total of 60 μL of elution buffer for the H3K4me3 and H3K9me3 ChIP assays. For each ChIP assay, the equivalent of 5 million cells was used for mESCs and 1 million cells for HEK 293T cells. These samples were analyzed by qPCR using primers and methods previously described (Table S1 ).15 (link) The antibodies used included G9a (Abcam #40542), H3K4me3 (Active Motif #39915), and H3K9me3 (Active Motif #39161).
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ChIP-qPCR Analysis of Histone Modifications
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Other organizations : University of North Carolina at Chapel Hill, North Carolina State University
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Variable analysis
independent variables
- Cell type (mESCs and HEK 293T cells)
- H3K4me3 and H3K9me3 levels as measured by ChIP-qPCR
- The ChIP-IT High Sensitivity kit (Active Motif #53040) was used according to the manufacturer's instructions
- Inputs were purified using a clean and concentrate column (Zymo Research #D5205)
- ChIP samples were eluted twice with 30 μL each for a total of 60 μL of elution buffer for the H3K4me3 and H3K9me3 ChIP assays
- The equivalent of 5 million cells was used for mESCs and 1 million cells for HEK 293T cells
- Primers and methods previously described (Table S1) were used for qPCR analysis
- Antibodies used included G9a (Abcam #40542), H3K4me3 (Active Motif #39915), and H3K9me3 (Active Motif #39161)
- Positive control: Not specified
- Negative control: Not specified
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