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Sample GSM8479334

Query DataSets for GSM8479334

Status

Public on Sep 18, 2024

Title

post 10wAIR_F1_RNAseq

Sample type

SRA

Source name

Lung

Organism

Mus musculus

Characteristics

tissue: Lung
Sex: female
cell type: Alveolar type II epithelial cells
treatment: 14 weeks filtered air

Extracted molecule

total RNA

Extraction protocol

Alveolar type II epithelial cells (Type II pneumocytes) were isolated according to published procedures. Briefly, the lungs were perfused with 10 mL cold phosphate buffered saline (PBS) before enzymatic digestion with 2 mL of dispase infused into the lung, after which they were removed and incubated in an additional 2 mL of dispase for one hour. The lungs were then manually disintegrated and the resulting cell suspension labeled with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32 (Thermo Fisher Scientific). Samples from 9 mice were pooled to include three sets of lungs per sample for a total of three samples for FACS separation. Type II pneumocytes were isolated by negative selection and thus identified as the unlabeled cell population. Type II pneumocytes were also gated as sideward scatter high (SSChigh) cell population which minimizes contamination with lymphoid cells by selecting cells with a higher granularity. The cells were separated by fluorescence activated cell sorting (FACS) by the University Flow Cytometry Resource at the University of Minnesota using a BD FACS Aria II P07800142 (BSL2) (BD Biosciences, San Jose, CA). Following isolation via FACS, Type II pneumocytes were pelleted by centrifugation. The samples were first centrifuged for 12 min at 200 x g and 4°C and the supernatant was removed, except for the bottom 1 mL, which was transferred to a 1.7 mL Eppendorf tube. To this tube, 500 µL of PBS was added, and the tube was centrifuged for 12 min at 200 x g at 4°C. The supernatant was removed while the bottom 100 μL was saved. One mL of PBS was added to the tube, and the sample was centrifuged for 12 min at 800 x g and 4°C. After this final centrifugation, all supernatant is removed, and the cell pellet was saved for downstream analyses. A portion of the cell pellet (1.25 x 105 – 5 x 105 cells) was set aside to isolate protein. The remaining sample was used to isolate DNA and RNA using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Hilden Germany) according to the man-ufacturer’s instructions.
Total RNA samples were converted to Illumina sequencing libraries using SMARTer Stranded Total RNA-Seq Kit – Pico Mammalian Input (Takara Bio USA, Mountain View CA).
Alveolar type II epithelial cells (Type II pneumocytes) were isolated according to published procedures. Briefly, the lungs were perfused with 10 mL cold phosphate buffered saline (PBS) before enzymatic digestion with 2 mL of dispase infused into the lung, after which they were removed and incubated in an additional 2 mL of dispase for one hour. The lungs were then manually disintegrated and the resulting cell suspension labeled with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32 (Thermo Fisher Scientific). Samples from 9 mice were pooled to include three sets of lungs per sample for a total of three samples for FACS separation. Type II pneumocytes were isolated by negative selection and thus identified as the unlabeled cell population. Type II pneumocytes were also gated as sideward scatter high (SSChigh) cell population which minimizes contamination with lymphoid cells by selecting cells with a higher granularity. The cells were separated by fluorescence activated cell sorting (FACS) by the University Flow Cytometry Resource at the University of Minnesota using a BD FACS Aria II P07800142 (BSL2) (BD Biosciences, San Jose, CA). Following isolation via FACS, Type II pneumocytes were pelleted by centrifugation. The samples were first centrifuged for 12 min at 200 x g and 4°C and the supernatant was removed, except for the bottom 1 mL, which was transferred to a 1.7 mL Eppendorf tube. To this tube, 500 µL of PBS was added, and the tube was centrifuged for 12 min at 200 x g at 4°C. The supernatant was removed while the bottom 100 μL was saved. One mL of PBS was added to the tube, and the sample was centrifuged for 12 min at 800 x g and 4°C. After this final centrifugation, all supernatant is removed, and the cell pellet was saved for downstream analyses. A portion of the cell pellet (1.25 x 105 – 5 x 105 cells) was set aside to isolate protein. The remaining sample was used to isolate DNA and RNA using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Hilden Germany) according to the man-ufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Project_017_Raw_Counts.txt.gz
Project_017_14w_FS_DEG_Test.csv.gz
14-f-air-1_S107

Data processing

Trimmomatic to remove low quality bases and adapter contamination
HISAT2 to map read pairs to the mm10 genome
Samtools to sort mapped reads by query name and filter by mapping quality
featureCounts to quantify gene expression
edgeR to analyze gene expression data
Assembly: mm10
Supplementary files format and content: CSV with the test results for differential gene expression. Contains Ensembl ID, base mean expression, log2(fold change), nominal P-values, and FDR values

Submission date

Aug 23, 2024

Last update date

Sep 18, 2024

Contact name

Natalia Tretyakova

E-mail(s)

[email protected]

Organization name

University of Minnesota

Department

Medicinal Chemistry