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Status |
Public on Jan 23, 2018 |
Title |
L-DKO DP #1 |
Sample type |
SRA |
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Source name |
sorted cells from thymus
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Organism |
Mus musculus |
Characteristics |
genotype/variation: L-DKO (deficient in Id2 and Id3) age: 4-5 weeks tissue: thymus cell type: double-positive (DP) thymocyte sorting strategy: CD1dTet- 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 sort, total RNA was extracted using the RNAqeous kit (Ambion) according to manufacturer's protocol. RNA was assessed for quality using the Agilent Bioanalyzer RNA Pico chip. Ribosomal RNA was depleted using the RiboErase method from Kapa Biosystems. In short, 1ug of total RNA was hybridized with 1ug of hybridization oligos tiling the 18s, 28s, 5.8s and mitochondrial rRNA sequences. Each sample was then RNaseH treated to degrade complementary rRNA sequence. The product was cleaned and purified using 2.2X AMPure beads (Agencourt). The cleaned product was DNase treated to degrade the DNA oligo mix. The remaining rRNA depleted samples were then purified using 2.2X AMPure XP beads. The Kapa Stranded RNA-Seq Kit was used to generate stranded Illumina sequencing libraries (Kapa Biosystems). RNA was fragmented at 94°C for 6 minutes. Briefly, RNA was hybridized to random primers, followed by first-strand cDNA synthesis, second-strand cDNA synthesis with marking, A-tailing, ligation of Illumina paired-end adapters with 8 bp barcodes, and 9 cycles of PCR amplification. Reactions were purified with Agencourt AMPure XP beads where necessary. Libraries were multiplexed in equimolar amounts and sequenced as paired-end 50-bp (100 bp) reads on an Illumina Hiseq 2500 V4 Sequencing System.
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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 12 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. FPKM gene expression measurements were log10 transformed after adding 1 to the expression values. Genome_build: mm10 Supplementary_files_format_and_content: Microsoft Excel file with FPKM values and fold change in knockout mice compared to wild type samples
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Submission date |
Nov 15, 2016 |
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) |
GSE89848 |
Id proteins suppress E2A-driven iNKT cell development prior to TCR selection [RNA-seq] |
GSE89849 |
Id proteins suppress E2A-driven iNKT cell development prior to TCR selection |
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Relations |
BioSample |
SAMN06017329 |
SRA |
SRX2348498 |
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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