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| Status |
Public on Jun 08, 2016 |
| Title |
Mus musculus WT-Th1 RA Med1 |
| Sample type |
SRA |
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| Source name |
In vitro differentiated Th1 cells, wild-type
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| Organism |
Mus musculus |
| Characteristics |
cell type: Th1
strain: C57BL/6
antibody: Bethyl Laboratories (A300-793A)
treatment/agent: PMA/ionomycin
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| Treatment protocol |
Human T cells were formaldehyde crosslinked on day 13 either before (unstimulated) or after (restimulated) treatment with PMA and ionomycin. For experiments testing the role of NF-κB, cells were restimulated for 5 hours with PMA/ionomycin in the presence of 20 µM BAY 11-7082 (Calbiochem) or DMSO before fixation. Human T cells for H3K4me3 ChIP-seq, cells were activated for 4 hrs with 5 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (CN Biosciences) EL4 cells were formaldehyde crosslinked before and after stimulation with PMA (50 ng/ml) and ionomycin (1 µM) for 5 hours.
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| Growth protocol |
Naïve human CD4+ T-cells (CD4+CD45RA+CD45RO-CD25-CCR7+) were isolated by negative immunomagnetic selection (Miltenyi Biotec) followed by sorting using a FACSAria II (BD Bioscience). Cells were activated for 72 hours by plate bound anti-CD3 and anti-CD28 antibodies (2 µg/ml, BD Pharmingen) and were then cultured for 10 days with rhIL-2 (Biolegend, 10ng/ml). Conditions for T cell polarization 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 H3K4me3 ChIP-seq, CD4+ naive T-cells were differentiated into either Th1 or Th2 cells in vitro for 28 days. Naïve murine CD4+ T-cells (CD4+CD25-CD62LhighCD44low) were purified from pooled spleen and lymph node cell suspensions of WT and T-bet -/- mice by immunomagnetic selection (Miltenyi Biotec) followed by sorting. 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 cell polarization were: 20 ng/ml IL-12 (eBioscience) and 5 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. CCR5+ memory Th1 cells were enriched with CD4 microbeads (Miltenyi Biotech) and CCR5+ Th1 (anti-CCR5, BD Biosciences) effector memory cells isolated by flow cytometry, activated with plate-bound anti-CD3 and anti-CD28 for 4 days, and expanded in media containing IL-2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells 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 detergent. For RNA pol II ChIP, an alternative set of lysis and wash buffers were used (Rahl et al., 2010). 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 pre-incubated with 10 µg of purified antibody or, for the case of T-bet, 10 µl of purified serum (see Table S6). 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 RNase A, proteinase K and phenol:chloroform extraction followed by ethanol precipitation. H3K4me3 ChIP was performed on native chromatin. Chromatin was prepared by using the protocol of Feil and colleagues (http://www.epigenome-noe.net/researchtools/protocol.php?protid=2) with some minor modifications. Mono and dinucleosomal chromatin was recovered from nuclei treated with micrococcal nuclease (10U/µl for 7 mins) and chromatin quality assessed by agarose gel elecrophoresis and semi-quantitated using a nanodrop. 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 clean up with Qiagen PCR purification columns.
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. The libraries were quantified using a Qubit and Agilent bioanalyzer, pooled and subjected to 35 or 50 bp single-end read sequencing with an Illumina GAIIx or HiSeq 2500 sequencer.
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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
Reads (in fastq files) were filtered to remove adapters using fastq-mcf and for quality using seqkt and aligned to the human (hg19) or mouse genome (mm9) with Bowtie2 (default settings). Bigwig files for visualization in the UCSC genome browser were generated using a custom pipeline; duplicate reads were first removed, coverage calculated with genomeCoverageBed and tag density calculated in 10bp windows. unionBedGraphs was then used to subtract input (or total histone H3 for histone ChIPs) signals and bigwig files generated using bedGraphToBigWig. Regions of significant enrichment were identified using MACS version 1.4 (Zhang et al., 2008) using input or total histone H3 as background, with the setting --keep-dup=1. A p-value threshold of 10-7 was used, unless stated otherwise.
Genome_build: hg19/mm9
Supplementary_files_format_and_content: peaks.bed
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| Submission date |
Dec 03, 2015 |
| 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 |
GPL13112 |
| 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 |
SAMN04313041 |
| SRA |
SRX1460843 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1961557_150716_CE3_ChIP_Mm_WT-Th1_RA_Med1_QC_10-7_peaks.bed.gz |
27.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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