At eight-to-ten-weeks old, mice were infected with aerosolized M. tuberculosis strain Erdman using a custom-built CH Technologies system. Twenty-four hours after each aerosol run, 4-12 mice were euthanized by carbon dioxide, and the entire lungs homogenized in 5mL sterile phosphate buffered saline, and all homogenate plated onto OADC-supplemented 7H11 agar. After 3 weeks at 37°C, M. tuberculosis colony forming units were counted to determine the retained lung dose.
Growth protocol
Four-to-five-week old female Diversity Outbred mice (n=1009) from generations 15, 16, 21, 22, 34, 35, 37, and 42 were purchased from The Jackson Laboratory (Bar Harbor, ME) and group housed (n=5-7 mice per cage) on Innovive (San Diego, CA) or Allentown Inc (Allentown, NJ) ventilated, HEPA-filtered racks in the New England Regional Biosafety Laboratory (Tufts University, Cummings School of Veterinary Medicine, North Grafton, MA). The light cycle was 12 hours of light; 12 hours of dark. Disposable caging was purchased sterile and re-usable caging was autoclaved prior to use. All cages were lined with sterile corn-cob bedding and sterile paper nestlets (Scotts Pharma Solutions, Marlborough, MA). Cages were changed at least every other week. Mice were provided with sterile mouse chow (Envigo, Indianapolis, IA) and sterile, acidified water ad libidum.
Extracted molecule
total RNA
Extraction protocol
One lung lobe from each mouse was homogenized in TRIzol™, stored at -80°C, and RNA was extracted using PureLink RNA Mini Kits (Life Technologies, Carlsbad, CA).
Label
biotin
Label protocol
Biotin labeling was performed using the WT Plus reagent kit (Affymetrix, Santa Clara, CA) according to the manufacturer's protocol.
Hybridization protocol
The labeled, fragmented DNA was hybridized to the Affymetrix Mouse Gene 2.0 ST for 18 hours in a GeneChip Hybridization oven 640 at 45oC with rotation (60 rpm). The hybridized samples were washed and stained using an Affymetrix fluidics station 450.
Scan protocol
After staining, microarrays were immediately scanned using an Affymetrix GeneArray Scanner 3000 7G Plus.
Description
Gene expression data from homogenized lung tissue of Asymptomatic Controller animal 200.
Data processing
Raw Affymetrix CEL files were normalized to produce Entrez-Gene-identifier-specific expression values using the implementation of the Robust Multiarray Average (RMA) in the affy Bioconductor package (version 1.62.0), using R version 3.6.0 and the Brainarray mogene20stmmentrezgcdf R package (version 24.0.0). Expression values were averaged together by animal prior to analysis.
Submission date
May 03, 2024
Last update date
Jun 15, 2024
Contact name
Boston University Microarray and Sequencing Resource
