GSM4977227: SKNMC_eKD2_K4me3; Homo sapiens; ChIP-Seq
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 200 bp. Chromatin equivalent to 5×106 cells was isolated and incubated with 5 μg antibody overnight at 4 °C (H3-acetyl K27, H3K4me3, and IgG (Diagenode). ChIP DNA was isolated by washing and reversal of cross-linking. The eluted DNA was used for SYBRgreen qPCR. 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 75 bases (Cat.TG-160-2005).