Illumina HiSeq 2000 paired end sequencing; Reduced representation of bisulfite sequencing (RRBS) of murine T-cell acute lymphoblastic leukemia (T-ALL) cells to investigate DNA methylation profiling of lymphocyte deficient for Tet2 and mutated for DNMT3A
Reduced representation of bisulfite sequencing (RRBS) of murine T-cell acute lymphoblastic leukemia (T-ALL) cells to investigate DNA methylation 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 EpiTect bisulfite Kit (QIAGEN) according to manufacturer's protocol Allprep Kit (QIAGEN) according to manufacturer's protocol RRBS libraries were prepared as described previously (Gu et al, Nature Protocols, 2011) with minor modifications. Genomic DNA (50-200ng) was digested for 5 hours with MspI, end-repaired, A tailed and ligated with T4 DNA ligase (Fermentas) to methylated Illumina adaptors. 150-400 bp fragments were gel-purified, bisulfite treated (EpiTect Bisulfite kit, Qiagen) and RRBS libraries were amplified by 15 cycles of PCR with PfUTurbo Cx hotstar DNA polymerase (Agilent) and indexed PE Illumina primers.