title: GSM1186422: MeDIP-seq-2 result; Mus musculus; MeDIP-Seq
library_construction_protocol: Mouse ES cells were crosslinked with formaldehyde treatment. The chromatin were isolated from 1 × 108 cells and fragmented to 200 to 400 bp by sonication. For MeDIP, we sonicated genomic DNA to produce random fragments ranging in size from 100 to 400 bp. DNA were repaired to blunt ends by T4 DNA polymerase and phosphorylated with T4 polynucleotide kinase using the END-IT kit (Epicentre). A single “A” base was added to 3' end with Klenow. Double-stranded adaptors were ligated to the fragments with DNA ligase. Ligation products between 200 and 600 bp were gel purified on 2% agarose to remove unligated adaptors and subjected to 20 PCR cycles. For MeDIP, The DNA ends were repaired using PNK and Klenow enzyme, followed by treatment with Taq polymerase to generate a protruding 3'-A base used for adaptor ligation. Following ligation of a pair of adaptors to the repaired ends, the fragments around 160 bp to 370 bp isolated from agarose gel.We used 1 ug of fragmented DNA for a standard MeDIP assay as described. Following denaturation at 95°C for 10 min, immunoprecipitation was carried at 4°C for 1 hr using 1 μg of monoclonal antibody against 5-methylcytidine  in a final volume of 500 μl IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100) and incubate at 4 C for 12 hours. And we added the mixture with 40 μl of Protein A/G beads (20421, Pierce) and Rabbit anti-mouse IgG (315-005-008, Jackson Immunoresearch) for 2 hr at 4°C and washed it for times with 700 μl of IP buffer. We then treated the beads with proteinase K for 2 hr at 50°C and recovered the methylated DNA by phenol-chloroform extraction followed by ethanol precipitation. Then the MeDIP-enriched DNA was amplified using the adaptor primers for 15 cycles and the fragments around 220 bp to 420 bp were isolated from agarose gel.
attributes.tag: GEO Accession
attributes.value: GSM1186422