GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4078082

Query DataSets for GSM4078082

Status

Public on Sep 01, 2020

Title

LungRep2

Sample type

SRA

Source name

lung-resident influenza-specific mouse CD4+ T cells

Organism

Mus musculus

Characteristics

tissue: Lung
cell type: CD4+ OT-II T cell

Treatment protocol

Lung tissue was mechanically disrupted using the gentleMACS tissue dissociator (Milteny Biotech). Tissue homogenates were then incubated for 30-60min at 37°C with digestion medium RPMI (Thermo Fisher) containing 10% fetal bovine serum (FBS) (Thermo Fisher), L-glutamate (Thermo Fisher), sodium pyruvate (Thermo Fisher), nonessential amino acids (Thermo Fisher), penicillin-streptomycin (Thermo Fisher), collagenase D (1 mg/ml, Roche), trypsin inhibitor (1 mg/ml, Thermo Fisher) and DNase I (0.1 mg/ml, Roche). After digestion, samples were once again mechanically disrupted using the gentleMACS dissociator, followed by a passing through a 100μm filter. After centrifugation, samples were depleted for red blood cells using ACK Lysing Buffer (Thermo Fisher) for 5min at room temperature. After washing and resuspending in RPMI containing 5% FBS, samples are filtered through a 70μm filter and kept on ice in the dark. Lymphocytes from spleens were isolated by first pressing the tissues through a 100μm filter with a plunger of a 1mL syringe to generate cell suspensions in RPMI containing 5% FBS. Spleen cell suspensions were further depleted for red blood cells using ACK Lysing Buffer as described above. Cells were then washed, resuspended, filtered through a 70μm filter and kept on ice until processing for flow cytometric analysis.

Extracted molecule

total RNA

Extraction protocol

After sorting using a BD Influx (BD Biosciences) sorter, cells were centrifuged and transferred into microcentrifuge tubes for extraction of RNA. Pellets of sorted cells were resuspended in 700ul of QIAzol Lysis Reagent (QIAGEN) and vortexed. Lysed samples were homogenated in the TissueLyser LT (QIAGEN) by shaking for 5min at 50Hz. Samples were left to rest for additional 5min at room temperature for complete dissociation of nucleoprotein complexes. Homogenized lysates were centrifuged through QIAshredder filters (QIAGEN). 100ul of gDNA eliminator solution was added. Samples received 140μl of chloroform followed by vortexing. Finally, samples were spun at 12,000 x g for 15min at 4°C. After centrifugation, the two phase solution contains RNA in the top transparent phase. The RNA phase was collected and transferred into a new microcentrifuge tube. An equivalent volume of 70% ethanol was added to the RNA-containg solution. The RNA/ethanol solution was transferred to an RNeasy MinElute spin column (QIAGEN) and washed with Buffer RWT and two rounds of Buffer RPE (QIAGEN). Following a final wash of the column with 80% ethanol, the RNA was eluted using RNase-Free water and stored at -80°C. Quality and quantity of total RNA was determined using the RNA 6000 Pico chip run on 2100 Bioanalyzer (Agilent Technologies).
Library preparation and RNA sequencing was performed by the Columbia University Genome Center. Total RNA samples were submitted and enriched for mRNA using a poly-A pull down strategy.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

FF025_S6_L007

Data processing

Read mapping to annotated transcriptome was done using kallisto (version 0.44). Mouse transcriptome Mus_musculus.GRCm38.cdna.all.fa.gz (Ensembl Relsease 94) was donwloaded from https://useast.ensembl.org/index.html
quant function of kallisto was used with arguments '--single -l 180 -s 20' for the single-end sequenced batch
Estimated counts generated with tximport(countsFromAbundance="lengthScaledTPM") for protein-coding genes
Genome_build: mm10
Supplementary_files_format_and_content: comma-separated text file includes estimated counts for each sample

Submission date

Sep 13, 2019

Last update date

Sep 01, 2020

Contact name

Donna L Farber

E-mail(s)

df2396@columbia.edu

Organization name

Columbia University

Department

Surgery