Chromatin immunoprecipitation (ChIP) was performed using an anti-YY1 antibody (Santa-Cruz; cat#sc-1703) as previously described [30 (link)]. In brief, chromatin collected from BMSCs was cross-linked using 1.0% formaldehyde and fragmented to about 200–1000 bp through sonication. Then 20 μg of chromatin was precleared with protein G-agarose beads (Santa Cruz) for 2 h at 4 °C, followed by immunoprecipitation with 3 μg of anti-YY1 antibody overnight at 4 °C. Precipitated samples were eluted and reverse cross-linked, and the ChIP DNA was analyzed using q-PCR. Four PCR primers were used: 5′- AGGTAGCGTTTGCTTTCTCACCCA -3′ (forward) and 5′-CAGCAATGCCAAGGGTAAACGGAA -3′ (reverse) (XIST promoter region); 5′- AGGGCTGCTCAGAAGTCTATCTCGGGGGCTC -3′ (forward) and 5′-GAGCCCCCGAGATAGACTTCTGAGCAGCCCT -3′ (reverse) (XIST promoter site1 region);
5′- CATTTAGGTCGTACAGGAACTCAAGTTCTTGGTGCGG -3′ (forward) and 5′- CGCACCAAGAACTTGAGTTCCTGTACGACCTAAATG -3′ (reverse) (XIST promoter site2 region); 5′- A ATGCTCTTGAATGTGTCTAAGTCATGTGACCTGCCC -3′ (forward) and 5′- GGGCAGGTCACATGACTTAGACACATTCAAGAGCAT -3′ (reverse) (XIST promoter site3 region).
5′- CATTTAGGTCGTACAGGAACTCAAGTTCTTGGTGCGG -3′ (forward) and 5′- CGCACCAAGAACTTGAGTTCCTGTACGACCTAAATG -3′ (reverse) (XIST promoter site2 region); 5′- A ATGCTCTTGAATGTGTCTAAGTCATGTGACCTGCCC -3′ (forward) and 5′- GGGCAGGTCACATGACTTAGACACATTCAAGAGCAT -3′ (reverse) (XIST promoter site3 region).