Eye AXBXA Illumina V6.2(Oct08) RankInv

    Accession number: GN210

    Summary:

FINAL RECOMMENDED AXB/BXA EYE DATA SET. The Eye AXBXA Illumina Illumina V6.2 (Oct08) data set provides estimates of mRNA expression for whole eyes of 28 strains of mice, including 26 AXB/BXA recombinant inbred strains, and two parental strains, A/J and C57BL/6J. All eye samples were obtained from normal adult control animals raised in a standard laboratory environment at the Jackson Laboratory. We used the Illumina Sentrix MouseWG-6 v2 BeadChip (despite the nomenclature, this is actually the third version of the Illumina Mouse-6 platform).

Users of these mouse eye data may also find the following complementary resources extremely useful:

1. NEIBank collection of ESTs and SAGE data
2. RetNet: the Retinal Information Network--tables of genes and loci causing inherited retinal diseases
3. Mouse Retina SAGE Library from the Cepko laboratory. This site provides extensive developmental data from as early as embryonic day E12.5.
4. Data sets on differential gene expression in anatomical compartments of the human eye from Pat Brown's lab. View expression signatures for different ocular tissues using the geneXplorer 2.0.

    About the cases used to generate this set of data:

A total of 54 pooled whole eye samples were processed using approximately 10 Illumina Sentrix Mouse WG-6 v2 oligomer BeadChip slides. All 10 slides and a total of 54 samples passed stringent quality control and error checking. This particular data set was processed using the Illumina "Rank Invariant" protocol. Values were log2 transformed. Variance of each array was stabilized to 4 units (SD of 2 units) and recentered to a mean of 8. values range from a low of 6.3 (e.g., ILMN_1225143, no expression) to a high of about 19.7 for ILMN_2772482 (Crygd, extremely highly expressed). Data were entered by Arthur Centeno, Hongqiang Li, Robert W. Williams, and Lu Lu, October 1, 2008.

As a measure of data quality we often count the number of probes that are associated with LOD scores of greater than 10. In this data set, 255 probes have LRS values >46 (LOD >10). The maximum LOD score achieved in this data set is 27.7 for Zfp330 (LRS of 127.9 using ILMN_2825109).

The probe ILMN_2475156 can be used to check sex assignment. With three exceptions, all 28 strains are represented by one male sample and one female sample. The three exceptions are are follows: both AXB13/14 cases are males, BXA25 is represented by a single male sample or a mixed sex sample, and BXA11 is represented by a single female sample.

Legend: Bar chart of the expression of Xist probe ILMN_2475156 in the AXB/BXA eye data set. This probe is used to check sex. Strains represented by equal numbers of male and female arrays (usually one of each) should have intermediate values and a high error term. Strains represented only by males will have very low values (for example, AXB13/14 is represented by only one male) and strains represented by only females will have very high expression (for example, BXA11 is represented by only one female).

    About the strains used to generate this set of data:

The AXB/BXA genetic reference panel of recombinant inbred strains consists of just about 26 fully independent strains. All of these strains are fully inbred, many well beyond the 100th filial (F) generation of inbreeding. All of these strains were been genotyped at 13,377 SNPs in 2005 (Shifman et al., 2006).

    About the animals and tissue used to generate this set of data:

AXB/BXA animals were obtained directly from The Jackson Laboratory. Animals were housed at UTHSC before sacrifice. Mice were killed by cervical dislocation and eyes and brains were removed and placed in RNAlater.

Animals used in this study were between 51 and 90 days of age (see Table 1 below). All animals were sacrifice between 9 AM and 5 PM during the light phase.

Sample Processing: Samples were processed by Lu Lu and colleagues in the Illumina Core at UTHSC between August 2008 and September 2008. All processing steps were performed by Dr. David Li. In brief, RNA purity was evaluated using the 260/280 nm absorbance ratio, and values had to be greater than 1.8 to pass our quality control (QC). The majority of samples had values between 1.9 and 2.1. RNA integrity was assessed using the Agilent Bioanalyzer 2100. The standard Eberwine T7 polymerase method was used to catalyze the synthesis of cDNA template from polyA-tailed RNA using the Ambion/Illumina (http://www.ambion.com/catalog/CatNum.php?AMIL1791) TotalPrep RNA amplication kit (Cat#IL1791). The biotin labeled cRNA was then evaluated using both the 260/280 ratio (values of 2.0-2.3 are acceptable) using a NanoDrop ND-1000 (http://www.nanodrop.com/nd-1000-overview.html). Those samples that passed QC steps (approximately 10% failed and new RNA samples had to be acquired and processed) were immediately used on BeadChips. The slides were hybridized and washed following standard Illumina protocols.

Replication and Sample Balance: We obtained a male sample pool and female sample pool from 25 of the 28 strains. Three strains are represented by samples from a single sex.

Experimental Design and Batch Structure: This data set consists arrays processed September 2008 and all arrays in this data set were processed using a single protocol by a single operator. Processing was supervised directly by Dr. Lu Lu. All samples were scanned on a single Illumina Beadstation housed in the Hamilton Eye Institute in September 2008. Details on sample assignment to slides and batches is provide in the table below.

    Data Table 1:

This table lists all arrays by order of strain (index) and includes data on tube ID, strain, age, sex, number of animals in each sample pool (pool size), slide ID, slide position (A through F), batch by slide number (1 or 2), and Source of animals.