Approximately 2ug of extracted genomic DNA from days 0, 4, 8, 10, 13, 17 and 29 were first mixed with 3.2pg of both unmethylated Lambda bacteriophage and in vitro methylated T7 bacteriophage DNA. Addition of the bacteriophage DNA was used to control for bisulfite conversion efficiency. Samples were then bisulfite converted using the EpiTect kit from Qiagen per the manufacturer's instructions. Primers designed to amplify UMRs, LMR and FMR regions (88 in total, Supplemental Table 1) were distributed in 96 well plates and amplification was carried out using Amplitaq gold using the following thermocycler settings (all temperatures are in celcius and all incubation times are 30” unless specified): 1 cycle of 95⁰ for 9', 20 cycles of touchdown with 95⁰ melt, 55⁰-51⁰ annealing, and extension at 72⁰, followed by 36 cycles of 95⁰ melt, 51⁰ annealing and 72⁰ extension. The amplicon reactions were then mixed, run out on an agarose gel and DNA extracted. Libraries were then constructed using the NEBNext ChIP-seq Library Prep kit (#E6240) as per the manufacturers instructions, indexed, pooled and sequenced using the Illumina MiSeq platform in 250bp paired-end mode.
GEO Accession
GSM4146766