Illumina HiSeq 2000 paired end sequencing; Genome-wide 5-hydroxymethylcytosine (5hmC) profiling by hMeDIP-seq in primary human CD4+T-cells polarized towards Th1 and Th2 time-series
Genome-wide 5-hydroxymethylcytosine (5hmC) profiling by hMeDIP-seq in primary human CD4+T-cells polarized towards Th1 and Th2 time-series
Primary naive CD4+T-cells were obtained from healthy blood donors using magnetic beads (MACS). Cells were cultured in RPMI-1640 under Th1 or Th2 polarizing conditions using plate-bound anti-CD3 and soluble anti-CD28 in the presence of IL-12 (5ng/mL), IL-2 (10ng/mL) and anti-IL4 (5μg/mL) or IL-4 (10ng/mL), IL-2 (10ng/mL), anti-IFNy (5μg/mL) and anti-IL12 (5μg/mL) up to 5 days for Th1 and Th2, respectively. Samples were collected for analysis at 0 (naive), 1 and 5 days post stimulation. Genomic DNA was extracted using an AllPrep DNA/RNA mini kit (Qiagen) DNA was fragmented by sonication using a Bioruptor (6 x 15min 30s on/off, Diagenode) then subjected to end-repair, dA-tailing, and adaptor-ligation using the NEBNext DNA Library Prep Master Mix Set for Illumina (New England Biolabs). Samples were hydroxymethylated DNA immuno-precipitated (hMeDIP) as previously described (Nestor et al., Genome Res 2012). Briefly, DNA was denatured and incubated with 1μg antibody against 5hmC (ActiveMotif; #39769) overnight at 4°C. IP and input DNA was prepared using magnetic beads (Dynabeads Protein G; Invitrogen) and amplified using NEBNext Multiplex Oligos for Illumina (New England Biolabs). Samples were separated by electrophoresis on a 2% low melting point agarose gel and fragments between 100 – 400 bp were selected. DNA was purified using the QIAquick Gel Extraction Kit (QIAGEN).
Experimental Factor: 5hmC
immunoprecipitate
Experimental Factor: T-helper 1 polarising
stimulus
Experimental Factor: 1
sampling time point