BB_NT
Homo sapiens
Protocols: 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).
ENA first public
2016-07-11
ENA last update
2016-05-03
External Id
SAMEA3950621
INSDC center alias
Department of Clinical and Experimental Medicine, Linkoping University
INSDC center name
Department of Clinical and Experimental Medicine, Linkoping University
INSDC first public
2016-07-11T17:01:30Z
INSDC last update
2016-05-03T16:09:53Z
INSDC status
public
Submitter Id
E-MTAB-4686:BB_NT
broker name
ArrayExpress
cell type
CD4-positive helper T cell
common name
human
individual
BB
organism part
blood
sample name
E-MTAB-4686:BB_NT