GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4175004

Query DataSets for GSM4175004

Status

Public on Dec 21, 2020

Title

A2 pLN NKG2a/clowCD16-

Sample type

SRA

Source name

lymph node NK cell subset from SIV chronically infected AGM (SV093)

Organism

Chlorocebus sabaeus

Characteristics

time post infection: 240
tissue: axillary lymph node
cell type: NK cells
subset: NKG2a/c LOW CD16 neg

Treatment protocol

A 8-color panels was used to phenotype, surface stain, and sort NK cells from blood and pLN from 3 chronically infected AGM. Cells were first thawed in 20% fetal bovine serum-containing media supplemented with Benzonase nuclease, and counts and viabilities were performed (life Technologies). Cells were washed and stained with Aqua Live/Dead stain (Molecular Probes). Cells were washed and blocked using normal mouse IgG (Caltag). For the NKG2a/cHIGH/low CD16+/- and CXCR5+ phenotyping panel, PBMC and pLN were surface-stained for CD3 (SP34.2, dilution 1/10, BD), CD8 (BW135/80, dilution 1/20, Miltenyi), CD16 (3G8, dilution 1/20, Beckman Coulter, Inc.), NKG2a/c (Z199, dilution 1/20, Beckman Coulter, Inc.), CD20 (2H7, dilution 1/20, Biolegend), CD14 (M5E2,dilution 1/25, BD) and CXCR5(MU5BEE, dilution 1:20, eBioscience™). Post surface staining cells were washed, filtered, and CXCR5+, NKG2a/clowCD16-, NKG2a/cHIGHCD16-, NKG2a/clowCD16+ and NKG2a/cHIGHCD16+ NK cells sorted on a FACS ARIA II (BD Biosciences).

Extracted molecule

total RNA

Extraction protocol

Cells were directly collected in a lysis buffer (RLT plus) containing TCEP. RNA was isolated using the RNeasy Plus® Micro Kit (74034, Qiagen). RNA integrity was verified with the Agilent Bioanalyzer. RNA from the different NK subsets were treated for library preparation using the Truseq Stranded mRNA sample preparation kit (Illumina, San Diego, California) according to manufacturer’s instructions.
Directional libraries were prepared using the Smarter Stranded Total RNA-Seq kit- Pico Input Mammalian following the manufacturer’s instructions (Clontech 635005). The quality of all libraries was checked with the DNA-1000 kit (Agilent) on a 2100 Bioanalyzer and quantification was performed with Quant-It assays on a Qubit 3.0 fluorometer (Invitrogen). Clusters were generated for the resulting libraries, with Illumina HiSeq SR Cluster Kit v4 reagents. Sequencing was performed with the Illumina HiSeq 2500 system and HiSeq SBS kit v4 reagents. Runs were carried out over 100 cycles, including seven indexing cycles, to obtain 100-bp single-end reads. Sequencing data were processed with Illumina Pipeline software (Casava version 1.9).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

African green monkeys (Caribbean Chlorocebus sabaeus, AGM)
LN-0-093

Data processing

Reads were cleaned of adapter sequences and low-quality sequences using cutadapt version 1.11.Only sequences at least 25 nt in length were considered for further analysis.
STAR version 2.5.0a, with default parameters, was used for alignment on the reference genome
Genes were counted using featureCounts version 1.4.6-p3 from Subreads package (parameters: -t gene -g ID -s 1).
Count data were analyzed using R version 3.4.3 and the Bioconductor package DESeq2 version 1.18.1. The normalization and dispersion estimation were performed with DESeq2 using the default parameters and statistical tests for differential expression were performed applying the independent filtering algorithm. A generalized linear model including the monkey identifier as blocking factor was set in order to test for the differential expression between the biological conditions (à completer/modifier en function des résultats exploités). For each pairwise comparison, raw p-values were adjusted for multiple testing according to the Benjamini and Hochberg (BH) procedure and genes with an adjusted p-value lower than 0.05 were considered differentially expressed.
Genome_build: chlorocebus sabaeus, from Ensembl release 90
Supplementary_files_format_and_content: Matrix from DESseq2

Submission date

Nov 18, 2019

Last update date

Dec 21, 2020

Contact name

Rachel Legendre

E-mail(s)

rachel.legendre@pasteur.fr

Organization name

Institut Pasteur

Department

Research and Resource Center for Scientific Informatics