Illumina HiSeq 2500 sequencing; Identify trimethylation marks from embryonic calvaria cell gene promoter regions from maternal high fat or control diet dams using H3K27me3 ChIP-Seq.
Identify trimethylation marks from embryonic calvaria cell gene promoter regions from maternal high fat or control diet dams using H3K27me3 ChIP-Seq.
After 17 days mice were sacrificed. For the isolation of fetal calvarial cells, sequential collagenase digestion of calvarial tissue was performed, and cells from second and third digestion were collected and pooled. Briefly, DNA was crosslinked to protein using formaldehyde and immunoprecipitated using CHIP grade H3K27ac antibody. After immunoprecipitation, chromatin was purified from the mixture using Cell signaling procedure. Purified DNA was then eluted from chromatin using Cell signaling protocal. Purified immunoprecipitated and input DNA were utilized to prepare libraries for sequencing using NEBNext reagents. Briefly, 150 ng of DNA was used for end-filling, dA-tailing and ligation with Illumina's paired-end adapters using manufacturer's recommendations.  Adapter-ligated DNA was amplified using PCR and products around 200 – 350 bp were used for cluster generation. Quantification of the ChIP-seq libraries was done via Qubit dsDNA HS Assay kit.