Computational protocol: Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2

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Protocol publication

[…] DNA was converted with the EpiTect Bisulfite kit according to the supplier's manual (Qiagen). Bisulfite-conversion-based methylation PCR primers were designed with the program Methprimer http://www.urogene.org/methprimer/index.html and in the case of IGF2_DMR2 with Methyl Primer Express (ABI). Primer sequences and product sizes of the 4 fragments covering the H19 DMD (H19_DMD_1, H19_DMD_2, H19_DMD_3, H19_DMD_4), the IGF2 DMR1 (IGF2_DMR1) and the IGF2 DMR2 (IGF2_DMR2) is shown in Additional file Table S1. PCR was performed with the Multiplex PCR Master Mix and products from liver, skeletal muscle and kidney were cloned (TOPO TA Cloning Kit (Invitrogen). White colonies were picked diluted in 50 μl water and amplified with the illustra™ TempliPhi amplification kit (GE Healthcare) and sequenced on an ABI 3730 capillary sequencer (Applied Biosystems). Bisulfite sequencing analysis was performed with the programs BiQ Analyzer http://biq-analyzer.bioinf.mpi-sb.mpg.de/ and MethTools http://genome.fli-leibniz.de/methtools/. […]

Pipeline specifications

Software tools methPrimer, MethTools
Applications BS-seq analysis, Epigenome data visualization
Organisms Sus scrofa, Mus musculus
Diseases Disorders of Sex Development