GSM1439668: IgG control_5hmC_DIP; Mus musculus; MeDIP-Seq
Immature myeloid cells (GFP+, CD11b-, Gr1-, CD16/32+) were sorted from AML1-ETO cultures. Genomic DNA was isolated using the AllPrep DNA/RNA micro kit (Qiagen). 5hmC DNA Immunoprecipitation and sequencing (5hmC-DIP-seq) of genomic DNA was performed as previously described (Williams et al., 2011, Nature 473, 343–348.). In brief, genomic DNA was sonicated using the Bioruptor (Diagenode) to obtain fragments of approximately 200-500bp. Next, adaptors were ligated using the NEBNext® DNA sample Prep Master Mix according to manufacturer’s instructions (New England Biolabs) and resuspended in 1× IP buffer (10mM NaPhosphate pH 7.0, 0.14M NaCl, 0.5% Triton X-100). 1ug of adaptor ligated DNA was briefly denatured and incubated 4h using an IgG control antibody or a polyclonal rabbit antibody directed against 5hmC. Anti-Rabbit dynabeads (Invitrogen) were added and incubated for additional 2h before immunoprecipitation, washing and elution by proteinase K digestion. The eluted DNA was PCR-amplified using indexed multiplex primers for illumina sequencing (New England Biolabs), pooled and sequenced on HiSeq 2000.
GEO Accession
GSM1439668