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Sample GSM8479357

Query DataSets for GSM8479357

Status

Public on Sep 18, 2024

Title

10wCS_F3_oxRRBS

Sample type

SRA

Source name

Lung

Organism

Mus musculus

Characteristics

tissue: Lung
Sex: female
cell type: Alveolar type II epithelial cells
treatment: 10 weeks cigarette smoke

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA isolated from Type II alveolar epithelial cells of control and treated A/J mice was prepared for RRBS and oxo-RRBS using the Ovation RRBS Methyl-Seq System with TrueMethyl oxBS module (NuGEN, Redwood City, CA) according to the manufacturer’s protocol. Library amplification was optimized as directed using qPCR, and the libraries were amplified accordingly followed by Agencourt bead clean-up.
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

Bisulfite-Seq

Library source

genomic

Library selection

Reduced Representation

Instrument model

NextSeq 550

Description

Project_012_MeC_hmC_Counts.txt.gz
Project_016_MeC_DMR.bed.gz
Project_016_hmC_DMR.bed.gz
10cs_fs3_OXO_S12

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)

trety001@umn.edu

Organization name

University of Minnesota

Department

Medicinal Chemistry