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| Status |
Public on Apr 28, 2023 |
| Title |
M1_D3_K4me3_S15 |
| 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 |
ChIP-seq sample preparation was performed with Covaris kit (PN 520154). Naïve T cells were cultured in vitro under Th9 promoting conditions and treated with chemical cross-linking (1% formaldehyde) on days 0, 3, 4, and 8 (murine) or days 0, 5, 8, and 15 (human). At least 0.5 million cells were used for each immunoprecipitation. After chemical chromatin cross-linking (1% formaldehyde), cells were washed and frozen at -80C. Cells were resuspended in Lysis Buffer B (Covaris) with protease inhibitors, and DNA fragmentation was performed on a Covaris Sonicator (ME220) for 8 min to an average length of 200-700 bp. After sonication, cells were immunoprecipitated with anti-H3K27Ac (ab4729; Abcam), anti-H3K4Me1 (ab8895; Abcam), and anti-H3K4Me3 (ab8580; Abcam). Genomic DNA (input) was prepared by treating aliquots of chromatin with proteinase K, and heated for de-cross linking, followed by column-based DNA purification (28106, Qiagen). An aliquot of chromatin underwent immunoprecipitation, after which decrosslinking was performed as above.
After recovering purified DNA, 5ng or more of DNA was used to generate libraries using the NEBNext Ultra Directional II DNA sequencing kit per the manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Basecalls were performed using bcl2fastq v2.20.0.422
Reads of 50 bases were processed using the ccbr pipeliner tool (https://ccbr.github.io/pipeliner-docs/): adapter sequences were trimmed with cutadapt 1.18
Data were mapped to the mouse genome mm10 or human genome GRCh38 (hg38) using BWAmem 0.7.17. Uniquely mapped and nonredundant reads were generated using Picard 2.17.11 and used for downstream analysis.
The aligned files were converted to bam format using SAMtools 1.6, and bigwig files were created using deepTools 3.0.1.
For downstream analysis, peaks were called using MACS v2.1.1 using a FDR 0.01 and with the input sample for background correction and using the --broad setting
Assembly: mm10 (mouse), hg38 (human)
Supplementary files format and content: bedfile, broadPeak
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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) |
| GSE222908 |
ChIP-seq of in vitro differentiated naïve 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 |
SAMN32740044 |
| SRA |
SRX19035349 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6935079_M1_D3_K4me3_S15_peaks.bed.gz |
537.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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