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| Status |
Public on Dec 27, 2011 |
| Title |
H3K36me3 WT Th1 |
| Sample type |
SRA |
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| Source name |
primary CD4+ T cells from spleen and lymph nodes
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J (wild type)
genetic background: C57BL/6J
cell type: in vitro polarized T helper1 cells for 7 days
passages: FACS sorted naive CD4+T cells cultured in vitro for 7 days under Th1 condition and restimulated with plate-bound CD3/28 +IL12 for 2 hrs
chip antibody: H3K36me3
chip antibody manufacturer: Abcam
chip antibody catalog #: 9050
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| Treatment protocol |
After 7 day culture in vitro, cells were restimulated for 2hr with plate-bound anti-CD3 and anti-CD28 (10 ug/ml each) and IL-12 (10 ng/ml).
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| Growth protocol |
Control and STAT4-deficient or T-bet-deficient naïve CD4+CD44-CD62L+ T cells were isolated and sorted on the flow cytometer. Cells were cultured with plate-bound anti-CD3 and anti-CD28 (10 ug/ml each), IL-12 (10 ng/ml) and anti-IL-4 (10 ug/ml) for 3 days, followed by IL-2 (50 U/ml) and IL-12 (10 ng/ml) for 4 days (Th1 conditions).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were chemically cross linked by 1% formaldehyde (Tbet-ChIP-seq) or treated with MNase (histone-ChIP-seq). Cell lysates were made by appropriate sonication and protein-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired and the blunt, phosphorylated ends were treated with Taq polymerase and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was PCR amplified with Illumina primers for 18 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
DNase-seq: Cells were washed twice with PBS, pelleted and resuspended in ice-cold Buffer A (15mM Tris-Cl, pH 8.0, 15mM NaCl, 60mM KCl, 1mM EDTA, pH 8.0, 0.5mM EGTA, pH 8.0, 0.5mM spermidine). Nuclei were isolated by mixing with 2x lysis buffer (Buffer A containing 0.01% NP-40) and incubated on ice for 5 min. Nuclei was then pelleted and washed with Buffer A. DNaseI digestions (60-180 U/mL) were performed for 3 min at 37°C, then stop buffer (50mM Tris-Cl, pH 8.0, 100mM NaCl, 0.1% SDS, 100mM EDTA, pH 8.0, 10ug/mL RNase A) was added. Nuclei were incubated at 55°C for overnight and then proteinase K was added for 6 h at 55°C. DNA was purified by phenol chloroform extraction, then fragments of 100-500 bp were isolated by sucrose gradient centrifugation as described previously (Sabo et al., 2006). Purified DNA was made into a library for Illumina deep sequencing platform to perform DNase-seq. Briefly purified DNA was end repaired, ligated to the Illumina single read adaptors, amplified and further purified to the fraction of 220-250bp in size. The resulted libraries are sequenced using the Illumina Genome Analyser Iix. Only unique reads of 36 bp were mapped to the mouse genome (mm9) using SICER peak calling program (Wei et al., 2010).
ChIP-seq: Cells were chemically cross linked by 1% formaldehyde (Tbet-ChIP-seq) or treated with MNase (histone-ChIP-seq). Cell lysates were made by appropriate sonication and protein-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired and the blunt, phosphorylated ends were treated with Taq polymerase and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was PCR amplified with Illumina primers for 18 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Chromatin IP against H3K36me3
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| Data processing |
Alignment: Sequence reads were obtained and mapped to the mouse mm9 genomes using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained and read starts were summed in sliding windows of 200 bp (SICER) or 100 bp (CisGenome) to create summary windows.
Peaks: Peak detection was performed with SICER (Zang et al, 2009 Bioinformatics, 25:1952-1958) for epigenetics modifcations and CisGenome ( Nature Biotechnology, 26: 1293-1300) for Tbet
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| Submission date |
Nov 18, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuka Kanno |
| E-mail(s) |
[email protected]
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| Phone |
301-402-3008
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| Organization name |
NIH
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| Department |
NIAMS
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| Lab |
LCBS-MIIB
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| Street address |
10 Center Drive, Rm13C120
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892-1930 |
| Country |
USA |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE33802 |
Early T-helper 1 Differentiation Is Marked by A Follicular Helper-like Transition: Differential Roles of STAT4 and T-bet |
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| Relations |
| SRA |
SRX107333 |
| BioSample |
SAMN00760422 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM836119_C4_H3K36me3-WT-Th1.bedgraph.gz |
2.4 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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