Epityper

Genomic DNA was isolated from dHPC samples using a commercial kit (QIAamp DNA Micro Kit, Qiagen, Hilden, Germany) following the manufacturer’s instructions and sent to Varionostic GmbH (Ulm, Germany) for EpiTYPER^® (Agena Bioscience, CA, USA) methylation analyses. Genomic DNA was treated with bisulfite to convert non-methylated cytosine to uracil, resulting in methylation-dependent sequence variations of C to T. These C/T variations appear as G/A changes on the reverse strand, which result in a mass difference of 16 Da per CpG site and can subsequently be detected by the MassARRAY system. The treated DNA was amplified by PCR and treated with shrimp alkaline phosphatase to neutralize unincorporated dNTPs. Subsequently, in vitro RNA transcription was performed, followed by base-specific cleavage at uracil residues using RNase A. The resulting fragments differ in mass and size, depending on the sequence changes due to bisulfite treatment, and generate characteristic signal patterns, which were identified by MALDI-TOF mass spectrometry.

High-Resolution Quantitative Methylation Profiling with EpiTYPER^® and the MassARRAY^® System (Agena Bioscience, San Diego, California), was used to validate the results from the discovery phase. EpiDesigner software for genomic target selection and PCR primer design was used to select top CpG sites from the discovery study suitable to be analysed by EpiTYPER^®. Two measures were captured for each CpG site and then averaged to avoid off-measurements. Pairs with a standard deviation bigger than 10% were excluded as part of the quality control.

Methylation analysis targeted a previously characterized 448-bp region of interest (hg19 chrX: 43,515,544–43,515,991) within the MAOA promoter comprised of 16 CpGs spanning the first exon and part of the first intronic region (Checknita et al. 2015 (link)). Genomic DNA extracted from saliva was first bisulfite-treated using EZ DNA Methylation™ Kit (Zymo Research Corporation, Irvine, CA) and then assayed using Agena Bioscience’s EpiTYPER at Karolinska University Hospital Mutation Analysis Core Facility (MAF). Resulting data represented the percentage of methylation at each CpG to the nearest 0.5%. CpGs were denoted numerically based on their 5′–3′ position within the ROI based on the forward strand genomic sequence. To ensure optimal technical outcomes, an amplicon designed on the reverse strand covered CpGs 1–13 and another amplicon covering CpGs 13–16 was designed on the forward strand. For additional information regarding the EpiTYPER procedure and quality control, please refer to Online Resource 1.

One microgram of DNA was treated with bisulfite using the EpiTect Bisulfite Kit (Qiagen, Hilden, Germany). DNA methylation of CpG sites in the promoter region of the OXT gene (chr20: 3,052,266–3,053,162; hg19 build) was analyzed using EpiTYPER (MassARRAY system; Agena Biosciences., San Diego, CA, USA) according to the manufacturer’s instructions. Forward (aggaagagagTTTTTTTGTTTTATTTTAGTGGTTTAGG) and reverse (cagtaatacgactcactatagggagaaggctTCTTACCTCCCAAAAAACAATTCTA) primers corresponding to chr20:3,052,009–3,052,392 were designed using EpiDesigner (Agena Bioscience, San Diego, CA, USA), and the spectrum characteristics were validated with RSeqMeth [28 (link)]. Further details were described in a previous study [22 (link)] where SN and AKS contributed as co-authors.

Genomic DNA was isolated from mice liver and adipose tissue using a TIANamp Genomic DNA Kit (#DP304, TIANGEN, China). Bisulfite conversion of genomic DNA was performed using the EpiTect Fast Bisulfite Conversion Kit (#59824, Qiagen, Germany). The CpG island region was selected to analyze the CpG methylation level. Target-specific primer pairs to amplify bisulfite-treated genomic DNA were designed using the EpiDesigner tool (Agena Bioscience, Inc., San Diego, CA, USA). Primers were synthesized by Generay, and each reverse primer had a T7 promoter tag (5′-CAGTAATACGACTCACTATAGGGAGAAGGCT-3′) for transcription. The forward primer was tagged with a 10-mer (5′-AGGAAGAGAG-3′) to balance the melting temperature. The polymerase chain reaction (PCR)-amplified products were treated with shrimp alkaline phosphatase, and in vitro transcription and base-specific cleavage were performed simultaneously. The primers of GRB2 associated binding protein 2 (GAB2) were as follows, forward: aggaagagagTAGATGGATTTGGGTAGTGTAATTG; reverse: cagtaatacgactcactatagggagaaggctACAACCTCCCCTCCAACCAA, which results 354 bp fragments. The resulting DNA fragments were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and EpiTYPER (Agena Bioscience, San Diego, CA, USA) was used for the quantification of CpG methylation level.