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| Status |
Public on Jul 19, 2019 |
| Title |
120_CTCF_ChIP_WT_Th2_CTCF_rep2 |
| Sample type |
SRA |
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| Source name |
mouse in-vitro activated Th2 cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
culture condition: 6 days in vitro culture
genotype: WT
tissue: mouse in-vitro activated Th2 cells
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| Growth protocol |
CD4+ T cells were purified from splenocytes and lymph nodes of untreated 6- to 14-week-old mice by negative selection and magnetic separation (Miltenyi Biotec). To obtain naïve T cells, CD4+ T cells were sorted into CD4+CD62L+CD44−CD25− T cells by fluorescence activated cell sorting (FACS) using FACSAria Illu or FACSAria Fusion (BD). Naive CD4+ T cells were activated on a 24-well-plate at a density of 4 x 10^5 cells/ml by plate-bound anti-CD3 (10 μg/ml, clone: 145-2C11) and anti-CD28 (10 μg/ml, clone: 37.51) in cRPMI (RPMI with 10% (vol/vol) FCS, 2 mM glutamine, 100 IU/ml of penicillin, 0.1 mg/ml of streptomycin and 20 mM HEPES buffer, pH 7.2–7.5, 1 mM sodium pyruvate, nonessential amino acids (all Thermo Fisher), and 2 μM β-mercaptoethanol (Sigma-Aldrich)) for 3 days with IL-12 (10 ng/ml, R&D Systems) and anti-IL-4 (10 µg/ml, 11B11) for Th1 cells or IL-4 (20 ng/ml, R&D Systems) and anti-IFN-γ (10 µg/ml, XMG1.2) for Th2. After 3 days, cells are split onto uncoated plates with fresh media and cytokines added along with IL-2 (100 U/ml, R&D Systems).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq, 10 million Th1 and Th2 cells cultured as indicated above were cross-linked for 10 min with 1% formaldehyde and harvested. Cells were lysed by sonication (Sonifier S-450 Digital Ultrasonic Cell Disruptor/Homogenizer, Branson Ultrasonics) in shearing buffer (50mM Tris-HCl pH 7.6, 0.2% Triton X) and immuno-precipitated using a polyclonal rabbit anti-mouse anti-CTCF antiserum (07-729, Millipore Sigma, 8 µl per assay). ChIP was performed overnight at 4 ˚C using Protein A Dynabeads (Thermo Fisher). Antibody-bound beads were washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE buffer (pH 8.0) containing 0.2% Triton X-100, and once with TE buffer (pH 8.0). DNA was released by incubating the beads at 65°C for 4 hrs in the presence of 0.3% SDS and 1 mg/mL Proteinase K. ChIP DNA was purified by a DNA clean and concentrator column (Zymo research). 80 ng of ChIPed DNA was subsequently used to prepare ChIP-Seq libraries using Ovation SP Ultralow DR Multiplex system (Nugen) following the manufacturer’s protocol.
Libraries were sequenced for 50 single read cycles on HiSeq 3000 following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
sample28_CTCF_ChIP20
lab data ID: 180629_JOO177_0146_AHVLT5BBXX/s08_2
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| Data processing |
V: CASAVA1.8.2/bcl2fastq1.8.4
alignment: SE reads were aligned to the mouse genome build mm9 with Bowtie 1.1.1
peak calling: Non-redundant and Uniquely mapped reads were used to call peaks with MACS1.4.2 using a p-value threshold of 1 x 10-5
Genome build: mm9
Processed data files format and content: Raw bw files
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| Submission date |
Jun 11, 2019 |
| Last update date |
Jul 19, 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 |
GPL21493 |
| Series (1) |
| GSE132531 |
The magnitude of IFN-g responses is fine-tuned by DNA architecture and the non-coding transcript of Ifng-as1 |
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| Relations |
| BioSample |
SAMN12018186 |
| SRA |
SRX6039132 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3873087_120_CTCF_ChIP_WT_Th2_CTCF_rep2.bw |
104.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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