H3k27me3 antibody

Detailed method is provided in Supplementary Methods. For immunoprecipitation, Oct4 antibody (Cell Signaling, 5677S) and SRF antibody (Active Motif, 61385, Santa Cruz, sc-335) were used at 6 μg/ChIP sample, H3K4me3 antibody (Millipore, 07-473), H3K27ac antibody (Abcam, Ab4729), and H3K27me3 antibody (Millipore, 07-449) were used at 1 μg/ChIP sample.

H3K27me3 chromatin immunoprecipitation (ChIP) was carried out using the ChIP kit (#26156, Thermo Fisher, United States) and according to Shen et al. (2014) (link). The pGCs were crosslinked by 1% paraformaldehyde and resuspended in PBS containing Halt cocktail. The supernatant was reserved following centrifuging after cell lysis. Then, 5 μL of lysed samples was used as input for analysis and 45 μL was used for further IP trials. Further, 10 μL aliquot of H3K27me3 antibody (#07-449, Millipore, Germany), 10 μL of the positive RNA polymerase II antibody, and 10 μL of negative IgG were added to each IP followed by incubation of the solutions overnight. Protein A agarose beads (10 μL) were washed with lysis buffer for three times and slowly mixed with the IP solution using a rotating mixer for 2 h. The mixture was washed with eluants and centrifuged at 4000 rpm for 5 min at 4°C. The ChIP eluant buffer was added to collect the binding DNA. The DNA was purified with a silica column and dissolved in an elution buffer. The DNA of the input, IgG, and H3K27me3 were used to amplify the H3K27me3 enriched region of MIR143. The RNA polymerase II IP and IgG IP were used to amplify the GAPDH enriched region. The PCR primers are listed in Supplementary Table 1. The qPCR of each group featured three replicates.

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.