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Status |
Public on Nov 01, 2017 |
Title |
WT neutrophils PBS rep4_miRNA |
Sample type |
RNA |
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Source name |
Lung neutrophils isolated from WT mice given PBS
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Organism |
Mus musculus |
Characteristics |
tissue: Lung cell type: neutrophils genotype: Wild type (WT) age: 6-12 weeks
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Treatment protocol |
Anesthetized mice received PBS or a suspension containing S. pneumoniae instilled intratracheally into the left lung at a dose of 2.3 µl/g mouse body weight. Neutrophils were isolated using MACS beads (Miltenyi, Auburn, CA) according to the manufacturer’s instructions. Briefly, single cell suspensions were prepared from dissected mouse lungs. The cells were washed with PBS containing 2 mM EDTA and 0.5% bovine serum albumin, and incubated with biotin-anti-Ly6G and magnetic beads coated with anti-biotin. The samples were passed through a column in a magnetic field, and cells bound to beads were collected for further analysis. Cells were kept at 4-8°C during staining and magnetic bead isolation.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated using miRNeasy kits from Qiagen (Valencia, CA, USA) according to the manufacturer's instructions.
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Label |
Cy3
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Label protocol |
100 ng of total RNA from each sample was labeled with Cyanine 3-pCp using Agilent’s miRNA Complete Labeling and Hyb Kit (p/n 5190-0456) according to the manufacturer's instructions.
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Hybridization protocol |
Labeled RNA was hybridized to Agilent SurePrint G3 Mouse miRNA 8X60K arrays for 20 hours at 55°C and 20 RPM. All reagents required for the labeling and hybridization steps were provided in Agilent’s miRNA Complete Labeling and Hyb Kit (p/n 5190-0456), and sample preparation and array processing were carried out according to the manufacturer's instructions.
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Scan protocol |
Slides were scanned immediately after washing on an Agilent Microarray scanner using the default settings for the array format (AgilentG3_miRNA for the 8x60K format).
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Description |
miR expression data from mouse lung neutrophils
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Data processing |
MicroRNA expression signals were extracted from microarray images using Agilent Feature Extraction (FE) software, and normalized using either LVSmiRNA or AgiMicroRna package from Bioconductor. LVSmiRNA uses the least-variant set of miRs to normalize between the arrays (Calza, S., Valentini, D. & Pawitan, Y. BMC Bioinformatics 9, 140, doi:10.1186/1471-2105-9-140 (2008)), while AgiMicroRna uses RMA. Data analyses were carried out on both LVS and RMA normalized log2 intensities in parallel. Both LVS and RMA normalized values are available on the series record.
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Submission date |
Apr 18, 2017 |
Last update date |
Nov 01, 2017 |
Contact name |
Claire M. Doerschuk |
Organization name |
University of North Carolina at Chapel Hill
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Department |
Marsico Lung Institute
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Street address |
Marsico Hall 7205 CB#7248
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City |
Chapel Hill |
State/province |
NC |
ZIP/Postal code |
27599-7248 |
Country |
USA |
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Platform ID |
GPL15547 |
Series (2) |
GSE97921 |
The mRNA and microRNA (miR) transcriptome of lung neutrophils during S. pneumoniae (SP) pneumonia in wild type (WT) mice [miRNA] |
GSE97922 |
The mRNA and microRNA (miR) transcriptome of lung neutrophils during S. pneumoniae pneumonia in wild type (WT) mice |
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