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Sample GSM2391491 Query DataSets for GSM2391491
Status Public on Jan 23, 2018
Title B6 NKT #2
Sample type SRA
Source name sorted cells from thymus
Organism Mus musculus
Characteristics genotype/variation: wild type
age: 4-5 weeks
tissue: thymus
cell type: Invariant Natural Killer T (iNKT) cells
sorting strategy: CD1dTet+ TCRβ+
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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
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
Submission date Nov 15, 2016
Last update date May 15, 2019
Contact name Yuan Zhuang
Phone 919-613-7824
Organization name Duke University
Department Immunology
Lab Zhuang lab
Street address 207 Research Drive
City Durham
State/province NC
ZIP/Postal code 27705
Country USA
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
BioSample SAMN06017314
SRA SRX2348501

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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