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Sample GSM7887500 Query DataSets for GSM7887500
Status Public on May 10, 2024
Title RNASeq STAT5B.48h.WT rep1
Sample type SRA
Source name spleen
Organism Mus musculus
Characteristics tissue: spleen
strain: C57BL/6J
cell type: freshly isolated CD8+ T cells
treatment group: STAT5B.48h.WT
Treatment protocol The purified CD8+ T cells were cultured in the presence of 500 U/ml of recombinant human IL-2, and then collected at 0, 4, 24, and 48 hr. The cells were washed with PBS once, lysed in TriReagent (Zymo Research, Irvine, CA), and total RNA was isolated using Direct-zol RNA MicroPrep Kit (Zymo Research, Irvine, CA).
Growth protocol Splenic CD8+ T cells were purified using the EasySep Mouse CD8+ T cell Isolation Kit (STEMCell Technologies, 19853) and cultured in complete RPMI 1640 medium either without or with 500 IU/ml recombinant human IL-2 (Biological Resources Branch Preclinical Biologics Repository, NCI).
Extracted molecule total RNA
Extraction protocol RNA-seq libraries were prepared using 100 ng of total RNA from splenic CD8+ T cells and the KAPA mRNA HyperPrep Kit (KR1352, KAPA Biosystems, Wilmington, MA), and each library was indexed using NEXflex DNA Barcodes-24 (BIOO Scientific, Austin, TX). Barcoded libraries were separated on 2% E-Gel, 250–400 bp fragments were purified, quantified on Qbit (Invitrogen), and equal amounts of each library were pooled and sequenced on an Illumina NovaSeq platform. For ChIP-seq libraries, freshly isolated splenic CD8+ T cells from Stat5a KI, Stat5b KI, and WT littermate mice were either not treated or treated with 500 IU/ml of IL-2 for 4 h, and cross-linked with 1% formaldehyde (methanol-free, Pierce, Rockford, IL) at room temperature for 10 min. After sonication at 30” ON and 30” OFF for 5 times using Bioruptor Pico (Diagenode), fragmented chromatin equivalent to 7 million cells were immunoprecipitated with control rabbit IgG (3900S, Cell Signaling Technology) or anti-STAT5A/B antibody (Ab194898, Abcam), and 20 μl of Magna ChIP Protein A + G Magnetic Beads (16–663, Millipore, Billerica MA). ChIP-Seq DNA libraries were prepared using KAPA Hyper Prep Kit (KR0961, KAPA Biosystems, Wilmington, MA) and NEXflex DNA Barcodes-24 (NOVA- 514103, BIOO Scientific, Austin, TX), and the indexed libraries were pooled and sequenced on an Illumina NovaSeq platform.
After quantification by Qubit (Invitrogen), barcoded samples were mixed and sequenced on an Illumina NovaSeq system.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Data processing Sequenced reads were aligned to the mm10 genome assembly using Bowtie 2.2.6 and Tophat 2.0.11
Aligned reads are converted to BAM files using samtools v0.1.8. BAM files are subsequently converted to BED files using bedtools v2.25.0. MACS is used to call peaks using the BED files. Unpublished python scripts were used to calculate RPKM (reads per kilobase per million reads) to normalize gene expression values for RNA-Seq data. R package edgeR was used to identify differentially expressed genes.
Assembly: mm10/GRCm38
Submission date Nov 08, 2023
Last update date May 10, 2024
Contact name Peng Li
Organization name NIH
Department NHLBI
Street address 9000 Rockville Pike
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
Platform ID GPL24247
Series (1)
GSE247343 Tyrosine phosphorylation of both STAT5A and STAT5B is necessary for maximal IL-2 signaling and its mitogenic action
BioSample SAMN38165399
SRA SRX22427806

Supplementary file Size Download File type/resource
GSM7887500_WJL2019_148.mm10.rpkm.gz 582.8 Kb (ftp)(http) RPKM
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