Chromatin immunoprecipitation (ChIP) qPCR and ChIP sequencing

ChIP was performed using iDeal ChIP-seq Kit for Transcription Factors (Diagenode, C01010170) according to manufacturer’s protocol. In brief, SK-N-MC and RD-ES cells of the control and knockdown groups were trypsinized, washed, and crosslinked with 1% formaldehyde in culture medium for 10 min at room temperature. Cross-linking was terminated by the addition of 1/10 volume 1.25 M glycine for 5 min at room temperature followed by cell lysis and sonication (Bioruptor, Diagenode), resulting in an average chromatin fragment size of 200bp. Chromatin equivalent to 5×10⁶ cells was isolated and incubated with 5 μg antibody overnight at 4°C (H3-acetyl K27, H3K4me3, H3K9me3, FLI1 and IgG (Diagenode). ChIP DNA was isolated by washing and reversal of cross-linking. The eluted DNA was used for SYBRgreen qPCR. The primer sequences used for ChIP-qPCR are provided in Table S6.

10ng of the ChIP DNA was used for ChIP sequencing library preparation following the protocol of TruSeq ChIP library preparation kit (Illumina, IP-202-1012). Briefly, A single “A” nucleotide was added to the 3′ ends of the blunt-ended ChIP DNA fragments and then ligated to a unique adapter. The ligation products were purified and selected at the size of 250–300bp by 4% NuSieve agarose gel (Lonza) electrophoresis. Size selected DNA was purified and PCR-amplified and quantitated with the Bioanalyzer 2100 (Agilent). Libraries were pooled and ran on Nextseq500 platform (Illumina Inc.) with high throughput single-end reads of 75bases (Cat.TG-160-2005).