GSM7664318: Pat022; Homo sapiens; Bisulfite-Seq
A 100 mg sample of omental adipose tissue was obtained during Caesarean delivery and washed thoroughly in ice-cold PBS to remove any blood. Fifty milligram portions were then transferred into cryotubes, snap-frozen in liquid nitrogen and stored at -80°C until DNA/RNA extraction. Omental adipose tissue DNA was isolated using Qiagen blood and tissue kits. Bisulfite conversion was performed on 500 ng genomic DNA using the Ez-96 DNA Methylation-Lightning MagPrep kit (Zymo Research). One microlitre of bisulfite converted DNA was used for the first round of polymerase chain reaction (PCR) using FastStart PCR Master (Merck) with gene-specific primers carrying tags for the second PCR. Primers were designed with a maximum amplicon length of 400 bp (Masser et al., 2015). All primers were tested by gradient PCR to achieve optimal conditions for second round PCR. Quality control was performed using a Qubit dsDNA high sensitivity assay kit. A 1.5% agarose gel was used to detect primer dimers and by-products from first-round PCR amplicons. Amplicons were then purified using the bead-based Merck AMPure XP kit  according to the manufacturer's protocol. Five microlitres of each of seven 4 nM amplicons derived from the same participant's sample were pooled and sample-specific adapter primers (barcodes) were bound to these tag sequences in the second round PCR. Equimolar pooling of libraries was conducted prior to sequencing on the Illumina MiSeq instrument. Targeted bisulfite amplicon sequencing for regions of the following genes: LEP, POMC, NPY, TNF, SOCS3.