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- NIAAA INIA Hippocampus Illumina (Aug07) Robust Spline Normalization (RSN) with
-Background Correction Database
- Summary: -August 07 ILLUMINA Mouse-6 DATA SET RSN: The LXS Hippocampus Illumina Robust Spline Normalization with No Background correction data set provides estimates of mRNA expression in the hippocampus of 75 LXS recombinant inbred strains, the two parental strains, ILS/Ibg and ISS/Ibg (Inbred Long Sleep and Inbred Short Sleep strains from the Institute of Behavioral Genetics), and the two reciprocal F1 strains (ILSXISSF1, ISSXILSF1). All samples are from normal adult control animals raised in a standard laboratory environment. Subsequent data sets will provide estimates of mRNA expression following restraint stress, ethanol treatment, and stress followed by ethanol using many of the same strains (Lu Lu and colleagues). -A total of 240 pooled hippocampal samples were processed using 40 Illumina Sentrix Mouse-6 v 1.0 oligomer microarray BeadArray slides. Twenty-seven Mouse-6 slides and a total of 160 samples passed stringent quality control and error checking. We should note that this is our first experience using the Illumina platform and the initial set of 13 slides were not included. This particular data set was processed using the Illumina "Robust Spline Normalization with Background Correction" protocol. Values were log2 transformed and the current data range from 6.417 average (very low or no expression) to 24.169 (extremely high). -As a measure of data quality we often count the number of probes that are associated with LOD scores of greater than 10 (LRS > 46). In this Hippocampus Illumina (Aug 07) RSN data set, ### probes have LRS values >46. -In comparison, here are the yields of QTLs with LOD>10 for other closely related data sets: -
The LRS achieved in the different version of the LXS Hippocampus data for probe ILM103520706 (Disabled 1; Dab1) are as follow -
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-ABOUT THE HIPPOCAMPUS. The hippocampus is an important and intriguing part of the forebrain that is crucial in memory formation and retrieval. This region of the brain is particularly vulnerable to the effects of environmental stressors and is a key upstream modulator of the hypothalamic-pituitary-adrenal axis (the HPA). The hippocampus is also often affected in epilepsy, Alzheimer's disease, and schizophrenia. Unlike most other parts of the brain, the hippocampus contains a remarkable population of stems cells that continue to generate neurons and glial cells even in adult mammals (Kempermann, 2005). This genetic analysis of transcript expression in the hippocampus (dentate gyrus, CA1-CA3) is a joint effort of 14 investigators supported by numerous agencies described in the Acknowledgments section. -About the strains used to generate this set of data: -The LXS genetic reference panel of recombinant inbred strains consists of just over 77 strains. All of these strains have been inbred for more than 23 generations (F23). All strains have been genotyped at 13,377 SNPs. Thanks to the efforts of Dr. Timothy Wiltshire at the Genome Institute of the Novartis Research Foundation, the two parental strains have been genotyped at 156,551 SNPs. These genotypes are incorporated in the GeneNetwork SNP Browser. -Strains are currently available from Drs. Beth Bennett and Tom Johnson at the Institute of Behavioral Genetics (IBG) in Boulder Colorado. -About the animals and tissue used to generate this set of data: -All animals were raised at the IBG by Bennett and colleagues in an SPF facility. No cases were MHV positive. Mice were killed by cervical dislocation. Whole brain dissections were performed at the IBG by Bennett and colleagues and shipped in RNAlater to Lu Lu and colleagues at UTHSC. Most hippocampal dissections (all were bilateral) were performed by Zhiping Jia. Cerebella, olfactory bulbs, and brain stems were also dissected and stored at -80 deg C using further use. Hippocampal samples are very close to complete (see Lu et al., 2001 but probably include variable amounts of fimbria and choroid plexus (see expression of transthyretin, Ttr, as a marker of choroid plexus). -A pool of dissected tissue from four hippocampi taken from two naive adults of the same strain, sex, and age was collected in one session and used to generate RNA samples. The great majority (75%) of animals were sacrificed between 9:30 AM and 11:30 AM. All animals were sacrificed between 9 AM and 5 PM during the light phase. All RNA samples were extracted at UTHSC by Zhiping Jia. -All animals used in this study were between 53 and 90 days of age (average of 72 days; see Table 1 below). -Sample Processing: Samples were processed by Lu Lu and colleagues in the Illumina Core at UTHSC between July 25 and Dec 20, 2006. All processing steps were performed by Feng Jiao. 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 (1-3% failed and new RNA samples had to be acquired and processed) were immediately used on the original Mouse-6 v 1.0 slide. 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 each strain. While all strains were orginally represented by matched male and female samples, one strain LXS34 consists of two female samples. Given the expression of Xist, we suspect that strain LXS114 is represented by two male pools (see figure at bottom of page). -Experimental Design and Batch Structure: This data set consists arrays processed in 13 groups over a five month period (July 2006 to Dec 2006). Most groups consisted of 12 samples. All arrays in this data set were processed using a single protocol by a single operator, Feng Yiao. Processing was supervised directly by Dr. Lu Lu. All samples were scanned on a single Illumina Beadstation housed in the Hamilton Eye Institute between July 28 and Dec 21, 2006. Details on sample assignment to slides and batches is provide in the table below. -Quality Control on Sex Labels: Sex of the samples was validated using sex-specific probe set: Xist (probe ILM106520068, also known as scl00213742.1_141-S). - |
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-Data Table 1: - -- ---This table lists all arrays by order of strain (index) and includes data on tube ID, strain, age, sex, F generation number, number of animals in each sample pool (pool size), slide ID, slide position (A through F), scan date, and scan batch. -- - --- |
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- Downloading all data: -All data links (right-most column above) will be available as soon as the global analysis of these data has been accepted for publication. Please see text on Data Sharing Policies, and Conditions and Limitations, and Contacts. Following publication, download a summary text file or Excel file of data. Please contact Dr. Lu Lu if you have any questions on the use of these open data. -About the array platform: -Illumina Sentrix Mouse-6 BeadArray Platform: The Mouse6 array consists of 46,116 unique probe sequences, each 50 nucleotides in length, that have been arrayed on glass slides using a novel bead technology. -Dunning M, Smith M, Thorne N, Tavare S (2006) beadarray: An R package to analyse Illumina BeadArrays. R News (the Newsletter of the P Project) 6:17-23. (see pages 17-23 of http://CRAN.R-project.org/doc/Rnews/Rnews_2006-5.pdf). -ANNOTATION: In spring of 2007, Robert W. Williams and Hongqiang Li reannotated the Illumina Mouse-6 array content. This new annotation is now incorporated into GeneNetwork. For 46166 probes on the Mouse 6 array platform (including control probes) we have identified 35975 NCBI Entrez Gene IDs; 26481 matched human Gene IDs; 23899 matched rat Gene IDs; 26883 NCBI HomoloGene IDs; and 12791 OMIM IDs. -Position data for the 50-mer Illumina Mouse-6 array were initially downloaded from Sanger at http://www.sanger.ac.uk/Users/avc/Illumina/Mouse-6_V1.gff.gz but we then updated all positions by BLAT analysis from mm6 positions to mm8 positions (Hongqiang Li). -About data processing: -This data set uses the LOESS with Variance Stabilizing Transform (VST) and Background correction from the lumi package downloaded from Bioconductor (http://www.bioconductor.org/). For the more detailed information, please see the lumi package documentation. -Data source acknowledgment: -Data were generated with funds to Lu Lu, Beth Bennett, Mike Miles, Melloni Cook from INIA. -ยท Lu Lu, M.D. -Grant Support: NIH U01AA13499, U24AA13513 (Lu Lu, PI) -About this text file: -INFO file prepared by Xusheng Wang, Oct 24, 2007. -Data set uploaded by Arthur Centeno, Aug 30, 2007. - |
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