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| Status |
Public on Jan 23, 2018 |
| Title |
pTαKO DP #2 (new) |
| Sample type |
SRA |
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| Source name |
sorted cells from thymus
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| Organism |
Mus musculus |
| Characteristics |
genotype/variation: pTA
age: 4-5 weeks
tissue: thymus
cell type: double-positive (DP) thymocyte
sorting strategy: TCRγδ- CD4+ CD8+
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| Extracted molecule |
total RNA |
| Extraction protocol |
Thymii were harvested from mice, and thymocytes were stained with appropriate antibodies for sorting. Following sorting in TRI reagent, RNA was extracted. RNA was assessed for quality using the Agilent Bioanalyzer RNA Pico chip. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads.
First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
RNA-sequencing reads were trimmed for removal of adapter sequences and low quality bases with Trimmomatic (version 0.32; adapters:2:20:7:1:true, leading:5, trailing:5, slidingwindow:4:10, minlen:21).
Reads were aligned with Tophat (version 2.0.9) to the mm10 transcriptome (Ensembl GRCm38), with remaining reads aligned to the mm10 genome.
Expression quantification was done using Cufflinks (version 2.2.0).
Log2 transformed FPKM (fragments per kilobase exon-model per million reads mapped) were used for downstream analyses. An FPKM gene expression matrix was generated for the samples.
Genes with low expression (FPKM< 1) in 50% of the samples, or with mean expression < 3 were filtered out as poorly expressed/covered genes. Samples found with expression > 1 in <10,000 genes were also filtered out as bad quality samples.
Genome_build: mm10
Supplementary_files_format_and_content: Microsoft Excel file with log2(FPKM) values and fold change in knockout mice compared to wild type samples
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| Submission date |
Jun 28, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuan Zhuang |
| E-mail(s) |
yuan.zhuang@duke.edu
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| Phone |
919-613-7824
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| Organization name |
Duke University
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| Department |
Immunology
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| Lab |
Zhuang lab
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| Street address |
207 Research Drive
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27705 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE89849 |
Id proteins suppress E2A-driven iNKT cell development prior to TCR selection |
| GSE100601 |
Id proteins suppress E2A-driven iNKT cell development prior to TCR selection [RNA-seq_new] |
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| Relations |
| BioSample |
SAMN07287417 |
| SRA |
SRX2964647 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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