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Sample GSM6757829 Query DataSets for GSM6757829
Status Public on Apr 15, 2024
Title CD4, CD8 double negative thymocytes in wildtype background. Mouse 1 [WT_DN_1]
Sample type SRA
 
Source name thymocytes
Organism Mus musculus
Characteristics genotype: wild type
strain background: C57BL/6
name stage_replicate: WT_DN_1
replicate id: 1
condition: WT_DN
stage (dn=cd4-cd8-,_dp=cd4+cd8+,_sp8=cd4-cd8+): DN
cell type: thymocytes
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
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
 
Submission date Nov 28, 2022
Last update date Apr 15, 2024
Contact name Tobias Suske
E-mail(s) tobias.suske@vetmeduni.ac.at
Organization name University of Veterinary Medicine
Department Functional Cancer Genomics
Lab Moriggl
Street address Veterinärplatz 1
City Vienna
State/province Vienna
ZIP/Postal code 1210
Country Austria
 
Platform ID GPL13112
Series (1)
GSE218858 Hyperactive STAT5 Hijacks T-Cell Receptor Signaling and Drives Immature T-Cell Acute Lymphoblastic Leukemia
Relations
BioSample SAMN31890541
SRA SRX18396411

Supplementary file Size Download File type/resource
GSM6757829_BSF_0512_H255CBBXY_3_WT_DN_1_S43615_unique_GeneCounts_2cols.tsv.gz 188.7 Kb (ftp)(http) TSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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