gender: Female age: 25 cell population: TCRαβ+ DN T cells
Treatment protocol
T cells were sorted into TCRαβ+ CD4+ T cells, TCRαβ+ CD8+ T cells, and TCRαβ+ DN T cell subsets by flow cytometry.
Growth protocol
n/a (isolated cell populations)
Extracted molecule
genomic DNA
Extraction protocol
Double negative T cells and autologous CD4+ and CD8+ T cells were isolated using the T cell isolation protocol (see overall design). RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) and RNeasy kits (Qiagen, Valencia, CA). RNA was then purified from DNA contaminants using a Turbo DNA-free kit (Ambion, Austin, TX) as per manufacturer’s instructions, and RNA concentration was measured with a Nanodrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE). Purified RNA was then used for the RT-PCR quantification of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B, relative to ACTB (β actin), using a ViaA 7 Real-Time PCR System (Applied Biosystems, Carlsbad, CA) and a Power SYBR Green RNA-to-CT 1-Step kit (Applied Biosystems, Carlsbad, CA) with 10μL reaction mixtures and PCR conditions suggested by the manufacturer.
The DNA methylation signature of human TCRαβ+CD4-CD8- double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response
