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| Status |
Public on Oct 19, 2021 |
| Title |
WT canonical Treg Rep3 |
| Sample type |
SRA |
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| Source name |
Sorted CD25+ FOXP3+ CD4+ CD3+ Tregs
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| Organism |
Mus musculus |
| Characteristics |
background strain: C57BL/6
genotype: WT
tissue: spleen
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with the Qiagen miRNeasy Mini kit. RNA quality and quantity were assessed with the Agilent 2100 Bioanalyzer. cDNA was prepared with the SMARTseq HT Kit (Takara Bio), using 1ng total RNA. Dual indexed sequencing libraries were prepared using the Nextera XT DNA Library Prep kit (Illumina), using 250pg cDNA.
Illumina TruSeq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
All data were processed and analyzed using the R programming language (Version 4.0.4), the RStudio interface (Version 1.4.1106), and Bioconductor
The first six samples (2 per group) were library prepped using the Bio-Rad SEQ-uoia library prep kit, and the last three samples (1 sample for each group, named "MM_13","MM_14", "MM_15") were library prepped using the SMART-Seq HT kit (Takara Bio). These nine samples were all sequenced in the same run. Still, the six samples (2 samples in each group) library prepped using the SEQ-uioa kit resulted in an insufficient number of reads, making a comparison between groups impossible.
We had to re-prep and sequence the first six samples again (named "WT_CD25posFOXP3pos_1","WT_CD25posFOXP3pos_2", "ITKCD25posFOXP3pos_1", "ITKCD25posFOXP3pos_2", "ITKCD25negFOXP3pos_1",”ITKCD25negFOXP3pos_2") using the SMART-seq HT kit. The batch effect from sequencing runs between the six samples re-prepped, and the three original samples were later removed.
Three groups were created: WT canonical, Itk-/- canonical, and Itk-/- noncanonical Tregs. Pseudoalignment with Kallisto (version 0.46.2) 26 was used to determine the transcript abundance of samples. Transcript per million (TPM) values were normalized, and the batch effect was removed by fitting to a linear model by empirical Bayes method with the Voom and Limma R packages 27. Bonferroni and Hochberg 28 adjusted p-value - False Discovery Rate (FDR) of ≤ 0.05 or FDR≤0.1 were used to describe the differential gene expression.
G:Profiler toolset, and g:GOSt tool was used for GO annotation analysis. C2 and C7 pathways collection from the Molecular Signatures Database (MSigDB) was utilized to perform Gene Set Enrichment Analysis (GSEA).
Genome_build: mm9
Supplementary_files_format_and_content: .tsv file, raw counts
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| Submission date |
Oct 05, 2021 |
| Last update date |
Oct 19, 2021 |
| Contact name |
Mobin Karimi |
| E-mail(s) |
karimim@upstate.edu
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| Phone |
3154642344
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| Organization name |
Suny Upstate Medical University
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| Department |
Microbiology/Immunology
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| Lab |
Karimi
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| Street address |
766 Irving Ave Suite 2281
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| City |
Syracuse |
| State/province |
NY |
| ZIP/Postal code |
13210 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE185327 |
Transcriptional analysis of noncanonical and canonical Tregs from ITK deficient mice (ITK KO) |
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| Relations |
| BioSample |
SAMN22060821 |
| SRA |
SRX12488121 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5610834_WT_CD25posFOXP3pos_2.tsv.gz |
1.6 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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