GSM9500708: HacaT cell, high glucose, C-FOS, ChIP; Homo sapiens; ChIP-Seq
The ChIP assays were performed using the SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, 9005) following the manufacturer's instructions. Briefly, HaCaT cells were cross-linked with 1% formaldehyde at room temperature for 10 minutes and then incubated in 2 ml of 10x glycine for 5 minutes. Cells were harvested in SDS lysis buffer and DNA fragments were digested to 150-900 bp fragments by Micrococcal Nuclease treatment. Then, samples were incubated overnight at 4°C with shaking with c-Fos antibody(Cat#31254S,Cell Signaling Technology) and normal IgG antibody as a control, followed by incubation with Magnetic Beads at 4°C with shaking for 2 hours the next day. After washing, chromatin was eluted from the beads using elution buffer and incubated with 5M NaCl and proteinase K at 65°C for 2 hours. The ChIP-seq library construction was performed as follows: The ChIP-enriched DNA fragments were first assessed for quality by quantification using a Qubit 4.0 Fluorometer and integrity analysis via agarose gel electrophoresis. Qualified DNA samples (20-100 ng) underwent end repair and dA-tailing (30°C for 20 min, 72°C for 20 min). Sequencing adapters were then ligated using Fast T4 DNA Ligase at 20°C for 15 min. The ligation product was purified with magnetic beads (0.6X ratio) to remove free adapters. The library was then amplified by 7 cycles of PCR (98°C for 10 sec, 60°C for 30 sec, 72°C for 30 sec) using index primers for enrichment, followed by another round of purification with magnetic beads (0.9X ratio).