GSM8560978: 5hmC_Normal_SM500; Mus musculus; OTHER
All prostate and tumor tissues were microdissected at necropsy, flash frozen in liquid nitrogen, and stored at -80°C Total DNA was purified from TRAMP tumor samples that were also evaluated through RNA-seq as there was sufficient tissue for both DNA and RNA extraction from the tumors. To obtain sufficient total DNA for normal mouse prostate hMeDIP-seq analysis, only RNA or DNA could be extracted from a single normal mouse prostate due to their small size. hMeDIP-seq was performed as a fee for service by Arraystar Inc. (Rockville, MD www.arraystar.com). Genomic DNA was sonicated to ~200 - 900bp with a Bioruptor sonicator (Diagenode). 800 ng of sonicated DNA was end-repaired, A-tailed, and ligated to single-end adapters following the standard Illumina genomic DNA protocol. Adaptor-ligated DNA was used for immunoprecipitation using a Rat monoclonal anti-5-hydroxymethylcytosine antibody (Diagenode). To recover the immunoprecipitated DNA fragments, 100 uL of protein G magnetic beads (Life Technologies) were added and incubated for an additional 2 hours at 4°C with agitation. hMeDIP and supernatant DNA were purified using Qiagen MinElute columns. 14 cycles of PCR were performed on 5 ?L of the immunoprecipitated DNA using the single-end Illumina PCR primers. Libraries were quality controlled by Agilent 2100 Bioanalyzer. The library was denatured with 0.1 M NaOH to generate single-stranded DNA molecules, and loaded onto channels of the flow cell at 8 pM concentration, amplified in situ using TruSeq Rapid SR Cluster Kit (#GD-402-4001, Illumina). Sequencing was carried out by running 100 cycles on Illumina HiSeq 2000 according to the manufacturer's instructions.