title: GSM2480759: B6_F_T_8 [Bisulfite-Seq]; Mus musculus; Bisulfite-Seq
library_construction_protocol: Naïve CD4+ T lymphocytes were purified from single-cell suspensions by MACS depletion of non-CD4+ T cells as well as regulatory T cells and TCRγ/δ+ T cells, using a cocktail of specific biotin conjugated antibodies against CD8a (Ly-2), CD45R (B220), CD49b (DX5), CD11b (Mac-1), Ter-119, CD25 and TCRγ/δ+ using the CD4+ CD62L+ T Cell Isolation Kit II (Miltenyi) to obtain an unlabeled pre-enriched CD4+ T cell fraction. Collected cells were suspended in PBS containing 2% FBS and Fc receptors were blocked with FcR blocking reagent for 10 minutes at 4°C (Miltenyi). Cells were further fluorescently stained with a combination of CD4-FITC (Miltenyi), CD44-APC clone IM7 (eBioscience), CD62L clone MEL-14 (BD Pharmingen), eFluor® 450 CD25 clone PC61.5 (eBioscience), for 10 minutes at 4°C, washed and then stained with DAPI (Life Technologies) to cell sort the live population of naïve CD4+, CD62Lhi, CD44lo, CD25- T lymphocytes using the MoFlo XDP system (Beckman Coulter). Samples were gated on single cells for doublet discrimination and non-viable cells were excluded using the viability dye 4', 6 – diamidine – 2 – phenylindole (DAPI). AllPrep DNA/RNA Mini Kit (Qiagen) was used to perform simultaneous isolation of RNA and DNA from 10^6 sorted naïve CD4+ T cells and resting B cells. triplicate 1µg samples of genomic DNA isolated from either naive CD4+ T cells or resting B cells were fragmented using a Covaris sonication system (Covaris S2). Following fragmentation, the DNA samples were spiked with synthetic fragments of DNA pre-labelled by PCR with 5mC and 5hmC used in the analysis step to determine the bisulfite conversion efficiency of cytosine forms (unmodified, 5mC and 5hmC). Libraries were prepared using the TruSeq DNA library preparation kit v2 (Illumina, FC-121-2001) according to manufacturer's instructions with some modifications. After ligation of TruSeq methylated adapters (ME-100-0010, Illumina) and AMPure® XP (Beckman) bead purification half of the sample was oxidized by treatment with KRuO4 (15 mM in 0.05 M NaOH) for 1 hr, performed as previously described
identifiers.namespace: GEO
identifiers.id: GSM2480759