Chromatin immunoprecipitation kit

Methyl Primer Express^TM v1.0 (Applied Biosystems, MA, USA) software was used to predict the methylation site of the CRABP2 promoter region and design relevant primers. Then, genomic DNA was extracted from the parental and OXA-resistant GC cells. Ultrasonication was performed for 10 sec, followed by a 30-s pause, which was repeated 4 times with 48 W of power. Genomic DNA was sheared into 200- to 1000-bp fragments under ultrasonic conditions. Then, according to the instructions of the Hydroxymethylation Quantitative Detection Kit (Abcam, Cambridge, UK), methylation-modified DNA fragments were obtained, and the difference in methylation modification between the two cell lines was detected by qRT–PCR. The parental and OXA-resistant GC cells were crosslinked with 1% formaldehyde, and the genomic DNA was sheared into 200- to 1000-bp fragments under the same ultrasonic conditions. Then, the CRABP2 promoter region was enriched using an anti-TET1 antibody (Abcam, MA, USA; ab272900) and was detected by the Chromatin Immunoprecipitation Kit (Abcam, MA, USA; ab500).

ChIP was carried out as Chromatin Immunoprecipitation kit (Abcam, Britain) described. Immunoprecipitation reactions were performed with anti-ZNF267 antibody (5 μg; Novus, USA), and IgG was as a control. Purified DNA was used for RT-PCR, and primers were designed specific to SGMS2 promoter. The fold enrichment was measured by comparing the Ct values of ZNF267-immunoprecipitated fraction to the IgG isotype fraction and normalized using the ΔCt formula.