title: GSM5105942: hMeDIP-seq-OL-2; Mus musculus; MeDIP-Seq
library_construction_protocol: For hMeDIP-seq, genomic DNA was sonicated to ~200-800bp 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 (FC-102-1002, Illumina). After agarose size-selection to remove unligated adapters, the adaptor-ligated DNA was used for immunoprecipitation (IP) with a mouse monoclonal anti-5-hydroxymethylcytosine antibody (Diagenode, C15200200). For this, DNA was heat-denatured at 94°C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μL primary antibody overnight at 4°C with rocking agitation in 400 μL IP buffer (0.5% BSA in PBS). To recover the immunoprecipitated DNA fragments, 20 μL of magnetic beads were added and incubated for an additional 2 hours at 4°C with agitation. After IP, a total of five washes were performed with ice-cold IP buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg/mL proteinase K for 2 hours at 65°C and then allowed to cool down to room temperature. DNA was then purified using Qiagen MinElute columns and eluted in 16 μL EB (Qiagen). 14 cycles of PCR were performed on 5 μL of the immunoprecipitated DNA using the single-end Illumina PCR primers. The resulting products were purified with Qiagen MinElute columns, after which a final size selection (300-1,000 bp) was performed by electrophoresis in 2% agarose. Libraries were quality controlled by Agilent 2100 Bioanalyzer. hMeDIP-seq