title: GSM2304712: ESS3.MeDIP; Homo sapiens; MeDIP-Seq
library_construction_protocol: DNA extraction used a QIAamp spin column method to yield a mean of 0.68 µg DNA/specimen for LCM sample. For bulk sample, 5-10ug DNA is isolated. For MeDIP, DNA isolated using the DNeasy Tissue kit (Qiagen, Valencia, CA) 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 and supernatant DNA were purified using Qiagen MinElute columns and eluted in 16 μl EB (Qiagen).Fifteen cycles of PCR were performed on 5 ul 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 (220-520bp) was performed by electrophoresis in DNA was eluted in 36 ul 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.