Tissues were fixed for 15 min at room temperature (RT) using 1% formaldehyde in phosphate buffered saline (PBS) containing protease inhibitors. Reversal of crosslinking was performed by incubation with 0.125 M glycine for 5 min at RT. Each pellet was re-suspended with lysis buffer (5 mM PIPES pH 8, 0.85 mM KCL, 0.5% Igepal) supplemented with 1X protease inhibitor cocktail, and sonicated for 30 min in 30 s ON/30 s OFF cycles (Bioruptor, Diagenode). The lysate was centrifuged at 14,000 g for 10 min and the supernatant transferred to a fresh tube. Optimal fragmentation was achieved by testing various sonication conditions on chromatin followed by DNA isolation and gel electrophoresis estimation of sonication efficiency. ChIP was performed using an AutoTrue MicroChIP kit (Diagenode #C01010140) on a SX-8G IP-Star Compact Automated System (Diagenode) as per the manufacturer’s instructions. Immunoprecipitation was performed using 3 μg of anti-H3K27me3 (Diagenode #C15410195) and non-specific IgG (Diagenode). Reverse crosslinking was performed with 5 M NaCl for 4 h at 65 °C. The immunoprecipitated DNA and input samples were purified using MicroChIP DiaPure columns (Diagenode #C03040001) and eluted with TE buffer. After ChIP, the crosslink was reversed and the DNAs were purified. To assess the quality and efficiency of the ChIP procedure, quantitative PCR was performed to assess the enrichment of known target genes. GAPDH, a housekeeping gene, was used as negative control for H3K27me3 ChIP. TSH2B gene, which is present in heterochromatin, was used as positive control for H3K27me3. TSH2B showed strong enrichment of H3K27me3 while GAPDH gene showed weak enrichment. Only samples with an enrichment of H3K27me3 above 5 were selected for ChIP-on-Chip analyses. Quantitative PCR was performed using SYBR Green Mix (Applied Biosystems #4309155) following the manufacturer’s instructions. The samples were amplified using an Applied Biosystems ABI Prism®7900 HT Real-Time PCR System (Applied Biosystems). PCR program was 95 °C for 3 min and 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The IP and input DNA were then subjected to microarray hybridization.