title: GSM1616760: MBD2 -/- Input DNA; Mus musculus; ChIP-Seq
library_construction_protocol: Bone Marrow Dendritic Cells (BMDCs) cells were crosslinked with 1% formaldehyde at room temperature for 10 min, followed by 5 min incubation with 125mM glycine. Chromatin was sheared by sonication and protein-DNA complexes were immunoprecipitated with an anti_H3K9,K14ac antibody (Milipore) and the DNA was subsequently extracted. ChIP DNA was end repaired by incubation at 20°C for 30 min with 3 U of T4 DNA Polymerase (NEB), 10 U of Polynucleotide Kinase (NEB), 2 U DNA Polymerase I Large (Klenow) fragment (NEB), 1x T4 DNA ligase reaction buffer (NEB) and 400 nM dNTPs. The enzymes were then heat inactivated at 75ºC for 20 min, after which the DNA was ethanol precipitated. A tail of 'A' bases was added to the 3’ ends of the DNA by incubation with 5 U Klenow Fragment (3’-5’ exo-; NEB), 200nM dATP and 1x buffer 2 (NEB) at 37°C for 30 min. The enzymes were heat inactivated and cleaned up as before .Barcoded Ilumina paired end adaptors were then ligated to the processed ChIP DNA by incubation with 300 U of T4 DNA ligase (NEB), 1x T4 DNA ligase buffer (NEB), 7.5% PEG-6000 and 2 pmol of annealed Illumina adaptors for 3 hr at room temperature. Ligated DNA was purified using MinElute PCR columns (Qiagen) and eluted in 10 μl water. Ligated DNA was amplified by 16-18 cycles of PCR with primers complementary to the adaptor sequences and Phusion 2x premix (Finnzymes). The DNA was purified using QIAquick PCR Purification columns (Qiagen).
identifiers.namespace: GEO
identifiers.id: GSM1616760