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| Status |
Public on Apr 15, 2024 |
| Title |
CD4, CD8 double negative in Rag deficient and Stat5aS710F background. Mouse 4 [R2S5a_DN_4] |
| Sample type |
SRA |
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| Source name |
thymocytes
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| Organism |
Mus musculus |
| Characteristics |
genotype: Stat5aS710F Rag2-/-
strain background: C57BL/6
name stage_replicate: R2S5_DN_4
replicate id: 4
condition: R2S5a_DN
stage (dn=cd4-cd8-,_dp=cd4+cd8+,_sp8=cd4-cd8+): DN
cell type: thymocytes
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| Extracted molecule |
total RNA |
| Extraction protocol |
DN, DP and SP8 T-cells were harvested from thymi by generating single-cell suspensions and FACS-sorting for viable Thy1.2-positive cells with respective CD4/CD8 expression patterns (Supplementary table). RNA was isolated from snap-frozen cell pellets using the DNA/RNA Allprep Kit (QIAGEN).
Library preparation and sequencing were performed at the Center for Molecular Medicine (CeMM), Vienna, Austria. Libraries were prepared using the NEBNext ®UltraTMRNA Library Prep Kit. Next, the samples were multiplexed and sequenced on an Ilumina Hisat2000 sequencer.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Quality control was performed using FastQC (version 0.11.8, Andrews S., 2010). Removal of low-quality bases and adapter trimming was achieved with trimmomatic (version 0.35, Bolger AM et al. 2014) followed by mapping using STAR aligner (version 2.7, Dobin A et al. 2013) with the GRCm38 genome (ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M25/GRCm38.primary_assembly.genome.fa.gz) and ensembl gene annotation release 93 (ftp.ensembl.org/pub/release-93/gtf/mus_musculus/Mus_musculus.GRCm38.93.gtf.gz, used in several SFBF61 projects). The genome for mapping with STAR was generated with the command: STAR --runMode genomeGenerate --genomeDir STAR93 –genomeFastaFiles GRCm38.primary_assembly.genome.fa –sjdbGTFfile Mus_musculus.GRCm38.93.gtf. For further analysis, only uniquely mapped reads were considered by using bamtools (Derek W et al.2011, Version 2.4.1) command: bamtools filter -tag NH:1 <mapped.bam> <filtered.bam>.
Counting features was achieved using GenomicAlignments package (Lawrence M et al. 2013) on the GRCm38.93.gtf annotation: reads mapping to exons with the tag “basic” were summarized using the GenomicAlignments::summarizeOverlaps() method. For RPKM calculations, the length of a gene was determined by summing up the width of the exons of a gene using the methods sum() and width(). Differential expression analysis contrasting each pair of conditions was performed using DESeq2 (Love MI et al. 2014, version 1.18.1).
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw counts for each Sample
Supplementary files format and content: Differential expression (DE) results using DESeq2 with single contrasts from RPKM-normalized values from the respective GeneCounts2cols.tsv files
Supplementary files format and content: all_DIFF_N642H_DE_result_tables.xlsx: focussed on contrasts along T cell differentiation and on STAT5BN642H
Supplementary files format and content: all_DIFF_S710F_DE_result_tables.xlsx: focussed on contrasts along T cell differentiation and on Stat5aS710F
Supplementary files format and content: all_GT_N642H_DE_result_tables.xlsx: focussed on contrasts across genotypes and on STAT5BN642H
Supplementary files format and content: all_GT_S710F_DE_result_tables.xlsx: focussed on contrasts across genotypes and on Stat5aS710F
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| Submission date |
Nov 28, 2022 |
| Last update date |
Apr 15, 2024 |
| Contact name |
Tobias Suske |
| E-mail(s) |
tobias.suske@vetmeduni.ac.at
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| Organization name |
University of Veterinary Medicine
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| Department |
Functional Cancer Genomics
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| Lab |
Moriggl
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| Street address |
Veterinärplatz 1
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| City |
Vienna |
| State/province |
Vienna |
| ZIP/Postal code |
1210 |
| Country |
Austria |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE218858 |
Hyperactive STAT5 Hijacks T-Cell Receptor Signaling and Drives Immature T-Cell Acute Lymphoblastic Leukemia |
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| Relations |
| BioSample |
SAMN31890531 |
| SRA |
SRX18396421 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6757839_BSF_0512_H255CBBXY_4_R2S5_DN_4_S43600_unique_GeneCounts_2cols.tsv.gz |
183.7 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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