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Status |
Public on May 10, 2024 |
Title |
RNASeq STAT5A.48h.WT rep1 |
Sample type |
SRA |
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Source name |
spleen
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Organism |
Mus musculus |
Characteristics |
tissue: spleen strain: C57BL/6J cell type: freshly isolated CD8+ T cells treatment group: STAT5A.48h.WT
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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).
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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).
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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.
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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 |
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
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Submission date |
Nov 08, 2023 |
Last update date |
May 10, 2024 |
Contact name |
Peng Li |
E-mail(s) |
peng.li@nih.gov
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Organization name |
NIH
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Department |
NHLBI
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Lab |
LMI
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Street address |
9000 Rockville Pike
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City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
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Platform ID |
GPL24247 |
Series (1) |
GSE247343 |
Tyrosine phosphorylation of both STAT5A and STAT5B is necessary for maximal IL-2 signaling and its mitogenic action |
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Relations |
BioSample |
SAMN38165423 |
SRA |
SRX22427798 |
Supplementary file |
Size |
Download |
File type/resource |
GSM7887476_WJL2019_124.mm10.rpkm.gz |
583.3 Kb |
(ftp)(http) |
RPKM |
SRA Run Selector |
Raw data are available in SRA |
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