GSM3669263: e_sgDnmt1_e4; Mus musculus; MBD-Seq
T cells were sorted based on CD8 and CD4 expression after 5 additional days of culture. DNA was isolated from T cells using phenol-chloroform extraction. For each sample, 100ng input of DNA was sheared with a Covaris LE220 instrument aiming for 450bp fragments. Sheared DNA was prepared with an EM-Seq kit (NEB) following the manufacturer's instructions. Briefly, the treatment includes a TET2-mediated oxidation of 5mCs to 5hmCs. This is followed by APOBEC cytosine deaminase treatment, resulting in conversion of unmethylated cytosines to uracil and their appearance as thymidines in the final sequencing data. Methylated and hydroxy methylated cytosines are indistinguishable from each other and are present as cytosines in the sequencing data. Unmethylated (Lambda) and fully CpG methylated (pUC19) DNA were spiked into each sample and used as internal control for conversion efficiencies. The final sequencing libraries were amplified with 4 Cycles of PCR and libraries were sequenced on Illumina HiSeqX targeting 30X coverage per sample.