* Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
* Normal versus disease
* Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
* OVA and Aspergillus model
* The number of hybridizations performed in the experiment.
* Total of 11 (6 for OVA and 5 for Asp)
* The type of reference used for the hybridizations, if any.
* Saline challenged mice
* Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
* We used Affymetrix oligonucleotide arrays where a single sample is hybridized to each chip
* Quality control steps taken: for example, replicates or dye swaps.
* URL of any supplemental websites or database accession numbers
* Excel sheet with average differences and absolute calls for all 11 chips (Table 1) and gene lists (Tables 2-4) on the Journal of Clinical Investigation web-site
Samples used, extract preparation and labeling:
* The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state.
* Lungs from male 10-12 week old mice challenged with saline or allergen.
* Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques.
* Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol.
* RNA was purified using Trizol™ reagent (per manufacurer’s recommendation) followed by phenol-chloroform extraction and ethanol precipitation
* Labeling protocol(s).
* Perform an in vitro transcription reaction using the ENZO BioArray HighYield RNA Transcript Labeling Kit (Affymetrix) according to manufacturer’s instructions.
* External controls (spikes).
* Control Oligonucleotide B2 (Affymetrix), 20X Eukaryotic Hybridization Controls (1.5 pM bioB, 5 pM bioC, 25 pM bioD, 100 pM cre) (Affymetrix)
Hybridization procedures and parameters:
* The protocol and conditions used during hybridization, blocking and washing.
* Create a hybridization cocktail for a single probe array that contains 0.067 &mgr;g/&mgr;L fragmented cRNA, 50 pM Control Oligonucleotide B2 (Affymetrix), 20X Eukaryotic Hybridization Controls (1.5 pM bioB, 5 pM bioC, 25 pM bioD, 100 pM cre) (Affymetrix), 0.1 mg/mL Herring Sperm DNA (Promega), 0.5 mg/mL Acetylated BSA (Invitrogen), and 1X Hybridization Buffer. Heat hybridization cocktail to 99°C for 5 minutes, to 45°C for 5 minutes, and spin at maximum speed in a microcentrifuge for 5 minutes. Fill probe array with 200 &mgr;L of 1X Hybridization Buffer. Incubate at 45°C for 10 minutes in the GeneChip Hybridization Oven 640 (Affymetrix) rotating at 60 rpm. Remove 1X Hybridization Buffer and fill probe array with 200 &mgr;L of the hybridization cocktail. Incubate at 45°C for 16 hrs in the Hybridization Oven rotating at 60 rpm.
Measurement data and specifications:
* The quantitations based on the images.
* The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
o Type of image analysis software used: specifications should be stated in the materials and methods.
o A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
o The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
o Data selection and transformation procedures.
o Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
We provide Excel data sheets with absolute calls and average differences for all tested genes, data we used to generate our significant lists. Finally, we provide our significant gene lists, after data selection and transformation. Details of scanning hardware, image analysis and data transformation are outlined in the methods section of the manuscript.
* General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known - often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
* Affymetrix U74Av2; synthesized in situ; part number 900343
* For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
* This is a commercially available array and can be viewed using MicroArray Suite software available form Affymetrix.
* For each reporter, its type (e.g., cDNA or oligonucleotide) should be given, along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database reference(s) and sequence (if available).
* For commercial arrays: a reference to the manufacturer should be provided, including a catalogue number and references to the manufacturer’s website if available.
* Affymetrix U74Av2; part number 900343; information available at www.affymetrix.com
* For non-commercial arrays, the following details should be provided:
o The source of the reporter molecules: for example, the cDNA or oligo collection used, with references.
o The method of reporter preparation.
o The spotting protocols used, including the array substrate, the spotting buffer, and any post-printing processing, including cross-linking.
o Any additional treatment performed prior to hybridization.