title: GSM1551778: Rat Blood MeDIP-seq; Rattus norvegicus; MeDIP-Seq
library_construction_protocol: For MeDIP, 5–15 μg DNA isolated using the DNeasy Tissue kit (Qiagen, Valencia, CA), than DNA was sonicated to ~100–500 bp with a Bioruptor sonicator (Diagenode). Sonicated DNA was end-repaired, A-tailed, and ligated to single-end adapters following the standard Illumina protocol. After agarose size-selection to remove unligated adapters, 2–5 μg of adaptor-ligated DNA was used for each immunoprecipitation using a mouse monoclonal anti-methylcytidine antibody (1 mg ml−1, Eurogentec. Catalog No. BI-MECY-0100). For this, DNA was heat-denatured at 95 °C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μl primary antibody per microgram of DNA overnight at 4 °C with rocking agitation in 500 μl immunoprecipitation buffer (10 mM sodium phosphate buffer, pH 7.0, 140 mM NaCl, 0.05% Triton X-100). To recover the immunoabsorbed DNA fragments, 4 μl of rabbit anti-mouse IgG secondary antibody (2.5 mg ml−1, Jackson Immunoresearch. Catalog No. 315-005-008) and 100 μl Protein A/G beads (Pierce Biotechnology. Catalog No. PI20421) were added and incubated for an additional 2 h at 4 °C with agitation. After immunoprecipitation a total of six immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg ml−1proteinase K for 2 h at 55 °C and then allowed to cool to room temperature. MeDIP DNA were purified using Qiagen MinElute columns and eluted in 16 μl EB (Qiagen).  Fifteen cycles of PCR were performed on 5 μl of the immunoprecipitated DNA using the single-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (220-520 bp) was performed by electrophoresis in DNA was eluted in 36 μl EB buffer using Qiagen QIAquick columns. After cleanup with Qiagen MinElute columns, each library was examined by spectrophotometry (Nanodrop, Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent).2% agarose. Libraries were quality controlled by spectrophotometry and Agilent DNA Bioanalyzer analysis.  For MRE:  DNA isolated using the DNeasy Tissue kit (Qiagen, Valencia, CA). Three parallel digests were performed (HpaII, AciI and Hin6I; Fermentas), each with 1μg of DNA. Five units of enzyme per microgram DNA were added and incubated at 37 °C in Fermentas Tango buffer for 3 h. Digested DNA was precipitated with sodium acetate and ethanol, and 500 ng of each digest were combined into one tube. Combined DNA was size-selected by electrophoresis on a 1% agarose TBE gel. A 100–300 bp gel slice was excised using a sterile scalpel and gel-purified using Qiagen QIAquick columns, eluting in 30 µl of Qiagen EB buffer. Library construction was performed using the Illumina Genomic DNA Sample Kit (Illumina) with single-end adapters, following the manufacturer’s instructions with the following changes. For the end repair reaction, T4 DNA polymerase and T4 polynucleotide kinase were excluded and the Klenow DNA polymerase was diluted 1:5 in water and 1 µl used per reaction. For single-end oligo adaptor ligation, adapters were diluted 1:10 in water and 1 μl used per reaction. Fifteen cycles of PCR were performed using the single-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (220-520 bp) was performed by electrophoresis in DNA was eluted in 36 μl EB buffer using Qiagen QIAquick columns. After cleanup with Qiagen MinElute columns, each library was examined by spectrophotometry (Nanodrop, Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent).2% agarose. Libraries were quality controlled by spectrophotometry and Agilent DNA Bioanalyzer analysis. 