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Sample GSM4775322

Query DataSets for GSM4775322

Status

Public on Apr 26, 2021

Title

Rep1 TH0 d1 TKO [RNA-Seq]

Sample type

SRA

Source name

sorted CD4+ naive T cells from lymph nodes and spleen, activated with anti-CD3+anti-CD28 Abs for 1 day

Organism

Mus musculus

Characteristics

age: 4,7 weks
genotype: Ikaros f/f CD4-Cre +

Growth protocol

Naive CD4+ T cells (DAPI-CD4+CD8-CD44-/lowCD25-NK1.1-TCRgd-) from lymph nodes and spleen were either left untreated (day 0) or cultured for 1 or 2 days under Th0 condition with plate-coated anti-CD3, anti-CD28 Abs (2μg/ml) in the presence of neutralizing anti-IL-4 and anti-IFNγ Abs (10μg/ml).

Extracted molecule

polyA RNA

Extraction protocol

FACS sorted naive CD4+ T cells were sorted from lymph nodes and spleen. Naive CD4+ T cells were directly used for the RNA-seq protocol for the day 0 condition, while the remaining naive CD4+ T cells were activated in Th0 culture condition for 1 or 2 days. At day 1 and 2, live cells (DAPI-) were sorted and used for the RNA-seq protocol. Total RNA extractions were performed using the RNeasy Plus Micro Kit (Qiagen 74034).
Full length cDNA were generated from 5 ng of total RNA using Clontech SMART-Seq v4 Ultra Low Input RNA kit for Sequencing (Takara Bio Europe, Saint Germain en Laye, France) according to manufacturer's instructions with 9 cycles of PCR for cDNA amplification by Seq-Amp polymerase. Six hundreds pg of pre-amplified cDNA were then used as input for Tn5 transposon tagmentation by the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina, San Diego, CA) followed by 12 cycles of library amplification. Following purification with Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France), the size and concentration of libraries were assessed by capillary electrophoreris.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

CRBD29

Data processing

Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Reads were preprocessed in order to remove adapters, polyA and low-quality sequences (Phred quality score below 20). After this preprocessing, reads shorter than 40 bases were discarded for further analysis. These preprocessing steps were performed using Cutadapt version 1.10.
Reads were mapped into the mm10 assembly of Mus musculus genome using STAR version 2.5.3a. The reads were aligned across exonic, intronic and intergenic genomic region using annotations from Ensembl 94.
Gene expression quantification was performed from uniquely aligned reads using Htseq-count version 0.6.1p1, with annotations from Ensembl version 94 and “union" mode. Only non-ambiguously assigned reads have been retained for further analyses.
In order to make these counts comparable between samples, read counts have been normalized across samples with the median-of-ratios method proposed by Anders and Huber.
Comparisons of interest were performed using the test for differential expression, proposed by Love et al. and implemented in the Bioconductor package DESeq2 version 1.16.1
Genome_build: mm10
Supplementary_files_format_and_content: A TSV (tab-separated values) file provides raw read counts and normalized read counts for each gene together with gene annotations and the p-value, adjusted p-value and log2 fold-change for each performed comparison. This file contains only genes with at least one read count in one sample.

Submission date

Sep 11, 2020

Last update date

Apr 26, 2021

Contact name

Tao YE

Organization name

IGBMC (CNRS/INSERM/UDS)

Street address

1 rue Laurent Fries

City

Illkirch