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| Status |
Public on Apr 01, 2020 |
| Title |
TH17(β) ATAC-seq rep2 |
| Sample type |
SRA |
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| Source name |
Nonpathogenic TH17 cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Nonpathogenic TH17 cells
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| Growth protocol |
Naïve CD4+ T cells were isolated from lymph nodes or spleen of mice using a CD4+ CD62L+ T cell isolation kit II (Miltenyi Biotec) according to the manufacturer’s instruction. Naïve CD4+ T cells were stimulated with plate-bound anti-CD3ε mAb (5 µg/ml) in the presence of anti-CD28 mAb (2 µg/ml) in a 48-well plate under neutral conditions (10 µg/ml anti–IL-4 mAb and 10 µg/ml anti–IFN-γ mAb), IL-6 conditions (10 ng/ml IL-6, 10 µg/ml anti–IL-4 mAb, and 10 µg/ml anti–IFN-γ mAb), TH17 (β) conditions (2 ng/ml TGF-β1, 20 ng/ml IL-6, 10 µg/ml anti–IL-4 mAb, and 10 µg/ml anti–IFN-γ mAb), TH17 (23) conditions (20 ng/ml IL-6, 20 ng/ml IL-1β, 20 ng/ml IL-23, 10 µg/ml anti–IL-4 mAb, and 10 µg/ml anti–IFN-γ mAb).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Naive CD4+ T cells from control and miR-21-/- mice were differentiated under pathogenic TH17 (23) differentiation condition. Total RNA was prepared from these cells using Trizol reagent (Invitrogen). RNA-seq libraries were prepared using a TruSeq Stranded Total RNA Library Prep Kit (Illumina). For GFP+ TH17 cells sorted from in vivo suspensions, 50,000 cells were resuspended in 700 ul Trizol reagent, total RNA was prepared by miRNeasy Micro Kit (Qiagen), RNA-seq libraries were prepared using SMART-Seq v4 Ultra Low Input RNA Kit (clontech). Sequencing was performed on an Illumina HiSeq X Ten System in a 150 bp/150 bp Paired end mode. ATAC-seq library preparations were performed as described(Buenrostro et al., 2013). In brief, 50,000 cells were washed in cold PBS and lysed. Transposition was performed at 37 °C for 30 min. After purification of the DNA with the MinElute PCR purification kit (Qiagen), DNA was amplified for 5 cycles. Additional PCR cycles were evaluated by real time PCR. Final product was cleaned by Ampure Beads at a 1.5× ratio. Libraries were sequenced on a HiSeq X Ten System in a 150 bp/150 bp Paired end mode.
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Data processing |
Basecalls performed using CASAVA version 1.4
ATAC-seq reads were trimmed using trim_galore 0.4.4_dev with default settings and then aligned to mm10 using bowtie2 with --end-to-end -X 2000. Reads with MAPQ < 10 were removed using samtools. Singletons are removed using in house Perl script. Duplicated reads were removed using samtools.
Filtered reads were sampled to the same reads number for comparing between samples using samtools. Differential chromatin accessible regions were identified using rgt-THOR version 0.1 with parameters --pvalue 0.05 --binsize 100 --step 50. Then significant differential peaks were further filtered according to the switch point from rank plot. Peaks were annotated to refGene annotation downloaded from UCSC Genome Browser using in house Perl script. Motifs were scanned using HOMER findMotifsGenome.pl from the top 1000 differential accessible peaks extended from the peak summit to +/- 100bp.
RNA-seq reads were mapped to mm10 with refGene annotation downloaded from UCSC Genome Browser using TopHat2 v2.1.0. Gene expression level were counted by htseq-count 0.11.0. Differential expressed genes were called by DESeq2.
Genome_build: mm10
Supplementary_files_format_and_content: BigWiig files were generated by rgt-THOR, scores representing the normalized ATAC-seq reads coverage. TAB-delimited text file included the raw reads count for each gene from RNA-seq sample.
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| Submission date |
Mar 04, 2019 |
| Last update date |
Apr 03, 2020 |
| Contact name |
Zhijun Han |
| E-mail(s) |
hangeneral@126.com
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| Organization name |
Southern University of Science and Technology
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| Street address |
No 1088,xueyuan Rd., Nanshan District
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| City |
Shenzhen |
| State/province |
Guangdong |
| ZIP/Postal code |
518055 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (1) |
| GSE127768 |
Inhibition of glycolysis in pathogenic TH17 cells through targeting a miR-21-Peli1-c-Rel pathway prevents autoimmunity |
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| Relations |
| BioSample |
SAMN11047535 |
| SRA |
SRX5459422 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3638391_TH17-b-3_normalized.bigWig |
86.3 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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