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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 01, 2011 |
| Title |
Th2S |
| Sample type |
SRA |
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| Source name |
Chromatin IP against BRG1, stimulated Th2
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| Organism |
Mus musculus |
| Characteristics |
strain: Balb/C
cell type: Th2 Stimulated CD4+ T helper cells
passages: differentiated in culture, single round, from Naïve cells
chip antibody: BRG (J1 antibody described in Genes Dev. 1996 Sep 1;10(17):2117-30, PubMed ID: 8804307)
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| Treatment protocol |
Stimulated cells: Effector cells were stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours.
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| Growth protocol |
Naïve CD4+ T cells were purified to 95% purity from the spleens and lymph nodes of 4-6 week old Balb/c mice (Taconic) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi).
Th1 cells: Naive, purified CD4+CD62L+ T cells cells were plated onto anti-CD3 (1μg/ml), anti-CD28 (2 μg/ml) coated plates at 1-2 x 106/ml in the presence of 1ng/ml IL-12, 10μg/ml anti-IL-4. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture.
Th2 cells: purified CD4+CD62L+ T cells cells were plated onto anti-CD3 (1μg/ml), anti-CD28 (2 μg/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10μg /ml anti-IFNγ. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture.
Th17 cells: Th17 cells were cultured using soluble anti-CD3 (4μg/ml), soluble anti-CD28 (1μg/ml), 10μg /ml both anti-IL-4 and anti-IFNγ, 100 ng/ml IL-6, 10ng/ml IL-1β and 1ng/ml TGF-β. The Th17 cultures were expanded in the presence of 10ng/ml IL-23 after three days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with formaldehyde, nuclei were treated with MNase, and sonicated with a bath sonicator (Bioruptor). BRG1-DNA complexes were isolated with antibody. The enriched DNA fragments or the sheared genomic DNA fragments (Input) were modified to construct a sequencing library according to the manufacturer’s protocol (Illumina/Solexa, San Diego, CA) with some modifications. Briefly, the ends of the fragments were repaired using T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase), and T4 polynucleotide kinase (PNK) and A’s were added to the 3’ end. Adapters were ligated to the DNA fragments and a 18-cycle PCR amplification was performed before size selection (250-300 bases) on a 2% agarose gel, followed by cluster generation and sequencing with Solexa/Illumina Genome Analyzer (GA-II).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Chromatin IP against BRG1
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| Data processing |
Alignment: The first 35 bases were aligned to the mouse mm9 genome using the Illumina Genome Analysis Pipeline v1.3.
Peaks: Peak detection was performed using CisGenome, 2 sample anaylsis, input as negative control, FDR 0.1, window size 100, boundary refinement and single strand filtering performed.
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| Submission date |
Aug 19, 2010 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
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| Department |
Laboratory of Genetics and Genomics
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| Lab |
Computational Biology & Genomics Core
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| Street address |
251 Bayview Blvd
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| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE23719 |
Dynamic BRG1 Recruitment During T Helper Differentiation And Activation Reveals Distal Regulatory Elements |
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| Relations |
| SRA |
SRX039653 |
| BioSample |
SAMN00199221 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM585297_Th2S.bedgraph.gz |
172.8 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM585297_Th2S_BRG1.bed.gz |
29.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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