Magnetic dynal beads

Chromatin immunoprecipitation (ChIP) was performed as previously described. Briefly, SKOV3 cells were rinsed with room temperature PBS before being crosslinked in a 1%formaldehyde solution. SKOV3 Cells were then harvested and homogenized in the presence of protease inhibitors before DNA was sonicated. Magnetic Dynal beads (Invitrogen, San Diego, CA, USA) combined with a mixture of antibodies (anti-CTCF, Cell Signaling Technology) was used to pull down CTCF overnight. Preimmune serum or IgG were used as negative controls. Sequencing libraries were generated for massive parallel sequencing using standard methods. Briefly, 500 ng of pulldown DNA was subjected to end repair, terminal adenylation, and adapter ligation before fragments ranging from, 400–500bp were isolated from a 2% E-gel (Invitrogen). After a standardized 12 cycle PCR, DNA quality was evaluated on a DNA 1000 Bioanalyzer chip (Agilent Technologies, Santa Clara, CA, USA) before being submitted for sequencing on an Illumina GAII (Illumina, Inc., San Diego, CA, USA). All ChIP-seq data is deposited in the Gene Expression Omnibus (GEO) database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE8545) and their accession number is: GSE85453.

Chromatin immunoprecipitation (ChIP)-PCR was performed as previously described [34 (link)]. In brief, 2 × 10⁶ OSCC cells were cross-linked with 1% formaldehyde and washed with PBS in the presence of protease inhibitors. Cells were homogenized and their chromatin was subjected to ChIP using magnetic Dynal beads (Invitrogen) and antibody against acetylated or trimethylated lysine 9 of histone H3 (H3K9) (Millipore, Temecula, CA). Fold-enrichment of amplified DNAs by ChIP was assessed using protocols as previously described [35 (link)]. Specific primers targeting the promoter region of TFPI-2 were as follows: R1-forward, 5′-GCAGGTCATTTCCGTCTAGCTT-3′ and R1-reverse, 5′-ACCTGCCTCCCAAACTTTCTC-3′; R2-forward, 5′-ACCACTTTCCCTCTCTTTTGCT-3′ and R2-reverse, 5′-TCGTAGTAGTAACGGAGAAGTAGGGC-3′.