GSM6590434: 24 month whole blood_B; Mus musculus; Bisulfite-Seq
tissues were lysed in 2% SDS and 25 mM EDTA overnight. Ammonium acetate (10M) was added to make the precipitate of SDS with proteins and was removed by centrifugation at 12,000 rpm. Next, DNA was precipitated using isopropanol, washed with 70% ethanol, and dissolved in dissolved in LTET (Tris 10 mM, EDTA, 0.1 mM + Tween 20,0.1% ) Biological duplicate samples of of all tissues were analyzed for DNA methylation changes by reduced representation bisulfite sequencing (RRBS) (Gu et al. Nature Protocols 2011, doi:10.1038/nprot.2010.190). We followed the NEB protocol for methylated adaptors. Briefly, 1 microgram of genomic DNA was spiked 100 picograms of lambda phage DNA as the unmethylated standard and digested with MspI. Ends of restriction fragments were filled in, 3'-dA tailed and methylated adaptors (NEB E7535) were ligated to the ends of restriction fragments. Bisulfite treatment using the Epitect kit (Qiagen) followed. Bisulfite-converted libraries were amplified using EpiMark Taq DNA polymerase (NEB) and primers with barcode indices. The libraries were pooled and sequenced at Fox Chase Cancer Center Genomics Facility/GENEWIZ/ Novogene on Illumina HiSeq2500 instrument using paired end reads of 80 bases (fox chase) and 150bp (Genewiz and Novogene). We used Bismark v0.18.1 to align the sequences to mm9 human genome assembly