GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3073382

Query DataSets for GSM3073382

Status

Public on Oct 08, 2018

Title

YAP cKO Naïve CD4+ T cells

Sample type

SRA

Source name

CD4+, CD62L+, CD25-

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue source: Lymph nodes and spleens
cell type: CD4+ T cells
cell markers: CD4+, CD62L+, CD25-
genotype: YAP cKO
treatment: Freshly isolated by FACS

Treatment protocol

Naïve CD4+ (CD62L+ CD25-) T cells and natural Treg (nTreg, CD4+ CD62L+/- CD25HIGH) cells were flow sorted from WT (wild type) and YAP cKO (YAP flox/flox,CD4-Cre+) mice. Freshly sorted naïve and nTreg cells (2×10^6 per sample) were washed with 1X PBS twice and immediately snap-frozen. Some nTregs were activated with 2µg/ml of plate-coated αCD3 and 2µg/ml of soluble αCD28 and IL-2 (100U/ml) for 24hrs before harvest, freezing, and eventual RNA extraction.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated by TRIZOL from naive CD4+ T cells, or natural Treg cells with or without the stimulation anti-CD3/CD28 for 48hr from wild type or YAP cKO mice.
Strand-specific RNA-seq libraries were prepared using TruSeq Stranded Total RNA LT Sample Prep Kit (with Ribo-Zero Gold, RS-122-2301, Illumina) from 322 ng of total RNA by following manufacturer protocols. Briefly, ribosomal RNA (rRNA) in both cytoplasm and mitochondria were depleted using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. After purification, the RNA was fragmented into small pieces using divalent cations under elevated temperature, which were transcribed into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. A single 'A' base was added to these cDNA fragments that were subsequently ligated with the adapter. The products were enriched with 12-cycle PCR. The concentration of final cDNA libraries in 30 ul ddH2O reached 24-27 ng/ul as determined on Qubit 2.0.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Sequencing was performed on Illumina Hiseq2000 at Beijing Genomics Institute with the type of paired-end, 100bp.
The quality of sequencing data were assessed by the software FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
The mapping to mouse reference genome of mm10 was conducted by TopHat.
Differentially-Expressed genes were called by Cuffdiff, and the normalized FPKM values were called by Cuffnorm.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file includes normalized FPKM values for all Samples.

Submission date

Apr 02, 2018

Last update date

Oct 08, 2018

Contact name

Jinsong Yan

E-mail(s)

yanjsdmu@dmu.edu.cn

Organization name

Dalian Medical University

Department

Institute of Cancer Stem Cell