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| Status |
Public on Jun 08, 2016 |
| Title |
Homo sapiens WT Th1 H3 for K27ac |
| Sample type |
SRA |
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| Source name |
In vitro differentiated Th1 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Th1
antibody: Abcam ab4729
treatment/agent: PMA/ionomycin
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| Treatment protocol |
Naïve human CD4+ T-cells (CD4+CD45RA+CD45RO-CD25-CCD7+) were isolated from healthy donors by negative immunomagnetic selection (Miltenyi Biotec) and activated for 72 hours by plate bound anti-CD3 and anti-CD28 antibodies (2 µg/ml, R&D) and then cultured for 10 days with rhIL-2 (Biolegend, 10 ng/ml). Conditions for T ell polarisation were: rhIL-12 (10 ng/ml, Biolegend) and anti-IL-4 (10 μg/ml, R&D) for Th1, rhIL-4 (10 ng/ml, Biolegend) and anti-IFN-γ (10 μg/ml, R&D) for Th2. For ChIP, cells were formaldehyde crosslinked on day 13 either before (unstimulated) or after (restimulated) treatment with anti-CD3 and anti-CD28 antibodies (2 μg/ml). For H3K4me3 ChIP-Seq, CD4+ naive T-cells were differentiated into either Th1 or Th2 cells in vitro for 28 days, following an established protocol. Cells were activated for 4 hrs with 5 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (CN Biosciences) and assessed for intracellular cytokine staining. Naïve murine CD4+ T-cells (CD4+CD25-CD62LhighCD44low) were purified by immunomagnetic selection (Miltenyi Biotec) from murine spleen and lymph nodes from WT and T-bet-/- mice (BALB/c background) or from WT and Gata3fl/fl x Tnfrsf4-Cre mice (C57BL/6 background). Cells were activated for 72 hours with plate-bound anti-CD3 and anti-CD28 monoclonal antibodies (both 2 μg/ml) and then cultured for 5 days in the presence of 20 ng/ml IL-2 (Biolegend). Conditions for T ell polarisation were: 20 ng/ml IL-12 (eBioscience) and 10 μg/ml anti-IL-4 (BioXCell) for Th1 or 20 ng/ml IL-4 (eBioscience) and 10 μg/ml anti-IFN-γ (BioXCell) for Th2. Cells were crosslinked on day 13 either before (unstimulaed) or after (restimulated) treatment with PMA (50 ng/ml) and ionomycin (1 μM) for 4 hours. EL4-GFP and EL4-T-bet cells were formaldehyde crosslinked before and after stimulation with PMA (50 ng/ml) and ionomycin (1 μM) for 4 hours.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Human Th1 and Th2 cells, pooled from multiple donors, were crosslinked by the addition of one-tenth volume of fresh 11% formaldehyde solution for 20 minutes at room temperature before the reaction was quenched by addition of glycine. Cells were rinsed twice with 1xPBS and flash frozen in liquid nitrogen. Cells were lysed with non-ionic detergent, nuclei washed and then lysed with ionic detegerent. Cells were sonicated on ice to solubilize and shear crosslinked DNA (24W for 10 x 30 second pulses using a Misonix Sonicator 3000). The resulting whole cell extract was cleared by centrifugation and then incubated overnight at 4°C with 100 µl of Dynal Protein G magnetic beads that had been preincubated with 10 ug of purified antibody or, for the case of T-bet, 10 ul of purified serum. Beads were washed 6 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C with occasional vortexing and crosslinks then reversed in IP and input DNA by overnight incubation at 65°C. IP and input DNA were then purified by treatment with RNAseA, proteinase K and phenol:chloroform extraction followed by ethanol precipitation. H3K4me3 ChIP was performed on native chromatin derived from ex vivo differentiated human Th1 and Th2 cells. Chromatin was prepared broadly using the protocol of Feil and colleagues with some minor modifications (http://www.epigenome-noe.net/researchtools/protocol.php?protid=2). Mono and dinucleosomal chromatin was recovered from nuclei treated with micrococcal nuclease (10U/µl for 7 mins). Chromatin quality was assessed by agarose gel elecrophoresis and semi-quantitated using a nanodrop. A second chromatin prep was also prepared from Th1 and Th2 cells, these cells were exposed to fomaldehyde in order to cross link protein to DNA. Nuclei from these cells were treated with MNase followed by sonication (6x20 second bursts at 40% amplitude using a Sonics VibraCell probe sonicator). ChIP for H3K4me3 and total H3 was performed with Protein G beads (Active Motif). DNA was purified by phenol/chloroform phase separation, ethanol precipitation, followed by Qiagen clean up.
Libraries were constructed from ChIP and input DNA by standard Illumina protocols, except that DNA in the range 150-350bp was gel-purified after PCR-amplification.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Basecalls performed using CASAVA
Polony identification, base calling and QC statistics were performed using GOAT and Bustard module
Genome_build: hg19/mm9
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| Submission date |
Oct 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard Jenner |
| E-mail(s) |
r.jenner@ucl.ac.uk
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| Organization name |
UCL Cancer Institute
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| Department |
Cancer Biology
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| Lab |
Regulatory Genomics
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| Street address |
72 Huntley Street
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| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE62482 |
T-bet activates poised Th1 genes through Mediator and the Super Elongation Complex [ChIP-Seq] |
| GSE62486 |
T-bet recruits P-TEFb to super-enhancers to regulate T helper cell differentiation |
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| Relations |
| BioSample |
SAMN03120332 |
| SRA |
SRX735294 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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