Illumina HiSeq 2000 sequencing; hMeDIP-sequencing of murine T-cell acute lymphoblastic leukemia (T-ALL) cells to investigate DNA hydroxymethylation profiling of lymphocyte deficient for Tet2 and mutated for DNMT3A
hMeDIP-sequencing of murine T-cell acute lymphoblastic leukemia (T-ALL) cells to investigate DNA hydroxymethylation profiling of lymphocyte deficient for Tet2 and mutated for DNMT3A
Cells were sorted using flow cytometry based on the expression of GFP and CD4/CD8 markers Allprep Kit (QIAGEN) according to manufacturer's protocol 5-hmC DNA Immunoprecipitation and sequencing (hMeDIP-seq) of genomic DNA was performed as previously described (Williams et al., 2011, Nature 473, 343348.). 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 manufacturers 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 antibody directed against 5-hmC. 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) and pooled.