ChIP-qPCR Protocol for H3K27me3

ChIP was performed as described previously^{50 (link)}. Approximately 1 × 10⁶ HSCs were incubated for 10 min at room temperature with 1% formaldehyde. After cross-linking, the reaction was quenched with 0.25 M glycine for 10 min at room temperature. Proteins were initially cross-linked to DNA and nuclei were then pelleted and sonicated to 200–500 bp fragments (Bioruptor, Diagenode). The cross-linked DNA was immunoprecipitated with H3K27me3 antibody (Millipore, USA) overnight at 4 °C with rotation. DNA-Antibody complexes were bound to ChIP beads, pulled down, washed and then eluted from beads. Following reversal of cross-linkage, purified DNA was used for Quantitative PCR using ChIP PCR primers that were purchased from IDT (MA, USA). Immunoprecipitation efficiency was calculated by normalizing sample C_T values against control IgG values and calculating ratios of sample C_T values relative to input values.