GSM6415370: Bone Marrow (Lin-, Sca1+, cKit+), Tet2-WT, vehicle, replicate 2; Mus musculus; Bisulfite-Seq
Bone marrow (BM) cells were isolated from each animal by flushing the bone 1x PBS 2% FBS. Red blood cells were lysed using ammonium chloride solution (Stemcell Tech). BM was FC blocked, then stained for lineage (CD3, CD4, CD8, B220, Gr-1, Ter-119, CD19, IgM, and CD127), Sca1, CD135, APC-conjugated lineage cocktail (described above), CD34, cKit, CD150, Fc Gamma (CD16/CD32), CD48, 7AAD and Annexin V. Live (7AAD-) cells were sorted for the following populations: LSK (Lin-Sca1+cKit+), CMP (Lin- Sca1- ckit+ CD34+ Fc Gamma-), and GMP (Lin- Sca1- ckit+ CD34+ Fc Gamma+). Approximately 500 cells per cell type/conditon were resuspended in RLT Plus buffer (Qiagen) and directly bisulfite converted using Zymo EZ Methylation Direct kit. Libraries were constructed using post-bisulfite adapter tagging method optimized for low input material (Clark et. al., Nature Protocols, 2017; doi:10.1038/nprot.2016.187). Libraries were sequenced on NovaSeq6000 instrument PE 100 bp.