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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 28, 2023 |
| Title |
mouse2_ATAC_s02 |
| Sample type |
SRA |
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| Source name |
In vitro differentiated Th9 (d3)
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: In vitro differentiated Th9 (d3)
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| Treatment protocol |
Murine naïve CD4+CD62L+ cells were isolated from spleen and lymph nodes using negative selection (Miltenyi) followed by flow cytometric sorting to >95% purity. Cells were cultured at a density of 0.5 x10^6 per mL and activated with plate bound aCD3Ɛ (10 µg/mL) and aCD28 (10 µg/mL) for 3 days with polarizing cytokine and antibodies to promote the differentiation of Th9 (10 µg/mLaIFN-g, 20 ng/mL mIL-4, 10ng/mL hIL-2, 5 ng/mL hTGF-β) for 3 days.
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| Growth protocol |
Complete IMDM (10% FBS, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 55 μM 2-Mercaptoethanol
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Omni-ATAC-seq sample preparation was performed as previously described. After flow cytometric sorting, 50,000 cells were lysed with 50 µl cold ATAC-Resuspension buffer (RSB) (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20) with 0.1% NP40, and 0.01% Digitonin on ice for 3 min. Lysis was stopped with 1 mL cold ATAC-RSB.
After centrifugation (500×g for 10 min), nuclei were resuspended in 50μl transposition reaction containing 2.5μl Tn5 transposase (FC-121–1030; Illumina) to tag and fragmentalize accessible chromatin. The reaction was incubated at 37°C, 400rpm for 30 min; DNA was then purified using a MinElute kit (QIAGEN) and amplified with 8–12 cycles of PCR based on the amplification curve. Samples were purified using a MinElute kit, sequenced on a NovaSeq 6000, paired end, 50 cycles
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Basecalls were performed using bcl2fastq v2.20.0.422
Redundant paired end (PE) reads were removed using fastquniq
PE reads of 50 bases were aligned to the mouse genome build mm10 or human build hg19 with Bowtie 0.12.8, following the guidelines presented by Buenrostro et al.
Customized python scripts were used to calculate fragment length of each pair of uniquely mapped PE reads for size distribution analysis, and to group uniquely mapped reads into bins of 0 to 175 bases and 180 to 250 bases, respectively.
UCSC Genome Browser viewable and normalized BigWig files were generated with the Hypergeometric Optimization of Motif EnRichment program (HOMER) version 4.8. Only one mapped read to each unique region of the genome that was less than 175 bp was kept and used in peak calling.
Regions of open chromatin were identified by MACS (version 1.4.2) using a p-value threshold of 1 × 10−5. Only regions called in both replicates were used in downstream analysis. Peak intensities (“tags” column) were normalized as tags per 10 million reads (RP10M) in the original library. Signal across all sites (i.e., all annotated genes and all accessible chromatin regions) was analyzed to eliminate potential bias by pre-selection.
Assembly: mm10 (mouse), hg19 (human)
Supplementary files format and content: bigWig, narrowPeak
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| Submission date |
Jan 14, 2023 |
| Last update date |
Apr 28, 2023 |
| Contact name |
Daniella Schwartz |
| E-mail(s) |
Daniella.Schwartz@pitt.edu
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| Organization name |
University of Pittsburgh
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| Street address |
200 Lothrop St
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15216 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE222907 |
ATAC-seq of in vitro differentiated naïve, Th1, and Th9 cells |
| GSE222910 |
ATAC-seq, ChIP-seq and RNA-seq of in vitro differentiated naïve, Th1, and Th9 cells |
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| Relations |
| BioSample |
SAMN32740059 |
| SRA |
SRX19035416 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6935064_mouse2_ATAC_s02_peaks.bw |
70.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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