NIAAA INIA Hippocampus Illumina (Aug07) Robust Spline Normalization with No Background Correction Database

Accession number: GN138

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 No Background Correction" protocol. Values were log2 transformed and the current data range from 6.481 average (very low or no expression) to 24.852 (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:

1. 1050 for Hippocampus Illumina (Aug07) LOESS
2. 1162 for Hippocampus Illumina (Aug07) LOESS_NB
3. 1129 for Hippocampus Illumina (Aug07) QUANT
4. 1176 for Hippocampus Illumina (Aug07) QUANT_NB
5. NO DATA for Hippocampus Illumina (Aug07) RSN
6. NO DATA for Hippocampus Illumina (Aug07) RSN_NB (THIS DATA SET)
7. 1183 for Hippocampus Illumina (May 07) RankInv
8. 1167 for Hippocampus Illumina (Oct06) Rank
9. 1170 for Hippocampus Illumina (Oct06) RankInv

The LRS achieved in the different version of the LXS Hippocampus data for probe ILM103520706 (Disabled 1; Dab1) are as follow

1. 338.4 for Hippocampus Illumina (Aug07) LOESS
2. 339.8 for Hippocampus Illumina (Aug07) LOESS_NB
3. 370.2 for Hippocampus Illumina (Aug07) QUANT
4. 363.5 for Hippocampus Illumina (Aug07) QUANT_NB
5. 374.8 for Hippocampus Illumina (Aug07) RSN
6. 363.0 for Hippocampus Illumina (Aug07) RSN_NB (THIS DATA SET)
7. 360.3 for Hippocampus Illumina (May 07) RankInv
8. 358.1 for Hippocampus Illumina (Oct06) Rank
9. 358.8 for Hippocampus Illumina (Oct06) RankInv

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).

    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.

index tube ID strain age sex gener- ation pool size slide ID slide position batch by slide scan batch 1 R2851H2 ILS 77 F >100 4 1523516003 B 1 1 2 R0595H2 ILS 71 F >100 2 1523516030 B 4 2 3 R2874H2 ILS 78 M >100 4 1523516011 B 2 1 4 R0585H2 ILS 65 M >100 2 1523516028 A 3 2 5 R3281H1 ILSXISSF1 90 F NA 3 1562224029 A 28 13 6 R2857H2 ISS 75 F >100 4 1523516011 A 2 1 7 R0589H2 ISS 73 F >100 2 1523516028 B 3 2 8 R2955H2 ISS 53 M >100 3 1523516003 A 1 1 9 R0578H2 ISS 67 M >100 2 1523516030 A 4 2 10 R3282H1 ISSXILSF1 97 M NA 3 1562224029 B 28 13 11 R2013H2 LXS10 84 F 27 2 1562224049 E 18 9 12 R1993H2 LXS10 78 M 27 2 1562224036 E 17 9 13 R1997H2 LXS100 70 F 25 2 1562224049 A 18 9 14 R1983H2 LXS100 77 M 25 2 1562224047 F 16 8 15 R2083H2 LXS101 85 F 28 2 1562224050 B 19 9 16 R2084H2 LXS101 85 M 28 2 1562224050 C 19 9 17 R2186H2 LXS102 73 F 29 2 1523516001 C 9 5 18 R2187H2 LXS102 73 M 29 2 1562224053 C 20 10 19 R2809H2 LXS103 72 F 26 2 1562224034 C 23 11 20 R2854H1 LXS103 69 M 26 2 1523516006 D 6 3 21 R2735H2 LXS107 78 F 26 2 1523516028 D 3 2 22 R2738H2 LXS107 78 M 26 2 1562224054 C 21 10 23 R2840H2 LXS110 67 F 25 2 1562224037 D 24 11 24 R2157H2 LXS110 75 M 27 2 1523516033 E 8 4 25 R2172H2 LXS112 72 F 27 2 1523516001 B 9 5 26 R2841H2 LXS112 84 M 27 2 1562224042 D 27 12 27 R2188H2 LXS114 72 F 28 2 1562224053 D 20 10 28 R2204H2 LXS114 72 M 28 2 1523516001 D 9 5 29 R2168H2 LXS115 80 F 27 2 1523516001 A 9 5 30 R2085H2 LXS115 71 M 27 2 1562224050 D 19 9 31 R2811H2 LXS117 72 F 28 2 1562224034 D 23 11 32 R2212H2 LXS117 74 M 27 2 1523516001 E 9 5 33 R1992H2 LXS122 86 F 26 2 1562224036 D 17 9 34 R2219H2 LXS122 72 M 26 2 1562224054 A 21 10 35 R2876H2 LXS123 87 F 28 2 1562224038 C 25 11 36 R2832H1 LXS123 77 M 27 2 1523516011 E 2 1 37 R2872H1 LXS124 83 F 28 2 1523516006 E 6 3 38 R2871H2 LXS124 85 M 28 2 1562224042 F 27 12 39 R1909H2 LXS13 79 F 25 2 1523516024 D 13 7 40 R1901H2 LXS13 81 M 26 2 1562224041 D 26 12 41 R2023H2 LXS14 86 F 25 2 1562224050 A 19 9 42 R1612H2 LXS14 85 M 26 2 1523516015 A 10 5 43 R1936H2 LXS16 81 F 25 2 1523516024 E 13 7 44 R1912H2 LXS16 81 M 25 2 1523516033 B 8 4 45 R1961H2 LXS19 66 F 26 2 1523516029 F 14 7 46 R1904H2 LXS19 66 M 26 2 1523516024 B 13 7 47 R1883H2 LXS2 71 F 27 2 1523516009 F 12 6 48 R2753H2 LXS2 74 M 28 2 1523516030 E 4 2 49 R1729H2 LXS22 72 F 27 2 1523516032 E 7 4 50 R2743H2 LXS22 79 M 27 2 1562224054 D 21 10 51 R1966H2 LXS23 73 F 26 2 1562224032 D 15 8 52 R1971H2 LXS23 73 M 26 2 1562224047 A 16 8 53 R2795H2 LXS24 74 F 26 2 1523516003 C 1 1 54 R1755H2 LXS24 56 M 25 2 1523516007 A 11 6 55 R1986H2 LXS25 75 F 27 2 1562224036 A 17 9 56 R2006H2 LXS25 75 M 27 2 1562224049 C 18 9 57 R2014H2 LXS26 82 F 25 2 1562224049 F 18 9 58 R2009H2 LXS26 82 M 26 2 1562224049 D 18 9 59 R2824H1 LXS28 67 F 28 2 1562224042 A 27 12 60 R1753H1 LXS28 72 M 27 2 1562224041 F 26 12 61 R2765H2 LXS3 63 F 27 2 1523516005 B 5 3 62 R1898H2 LXS3 81 M 26 2 1562224041 C 26 12 63 R2764H2 LXS31 73 F 29 2 1562224060 C 22 10 64 R1908H2 LXS31 80 M 28 2 1523516024 C 13 7 65 R2758H2 LXS32 70 F 26 2 1523516030 F 4 2 66 R1743H1 LXS32 67 M 25 2 1562224041 E 26 12 67 R2794H2 LXS34 75 F 27 2 1562224034 B 23 11 68 R2870H2 LXS34 78 F 27 2 1562224029 C 28 13 69 R2746H2 LXS35 80 F 22 2 1523516030 C 4 2 70 R2747H2 LXS35 81 M 22 2 1562224060 A 22 10 71 R1968H2 LXS36 76 F 25 2 1562224032 E 15 8 72 R1640H2 LXS36 76 M 25 2 1523516032 C 7 4 73 R2835H1 LXS38 67 F 27 2 1523516011 F 2 1 74 R2842H2 LXS38 67 M 27 2 1562224042 E 27 12 75 R2210H2 LXS39 81 F 28 2 1562224053 E 20 10 76 R2736H2 LXS39 75 M 27 2 1523516028 E 3 2 77 R1978H2 LXS41 78 F 26 2 1562224047 C 16 8 78 R1783H2 LXS41 56 M 26 2 1523516007 D 11 6 79 R2822H2 LXS42 66 F 27 2 1562224034 F 23 11 80 R2769H2 LXS42 70 M 27 2 1562224042 B 27 12 81 R1974H2 LXS43 84 F 26 2 1562224047 B 16 8 82 R1733H2 LXS43 72 M 26 2 1523516015 C 10 5 83 R1756H2 LXS46 56 F 25 2 1523516007 B 11 6 84 R1727H2 LXS46 70 M 25 2 1523516032 D 7 4 85 R1970H2 LXS48 72 F 27 2 1562224032 F 15 8 86 R1981H2 LXS48 72 M 27 2 1562224042 C 27 12 87 R1957H2 LXS49 72 F 25 2 1523516029 C 14 7 88 R2259H2 LXS49 72 M 25 2 1523516028 C 3 2 89 R2836H1 LXS5 68 F 28 2 1523516006 A 6 3 90 R2213H2 LXS5 80 M 27 2 1562224053 F 20 10 91 R2791H2 LXS50 68 F 27 2 1562224034 A 23 11 92 R1789H2 LXS50 57 M 26 2 1523516033 A 8 4 93 R1740H2 LXS51 68 F 27 2 1523516032 F 7 4 94 R1734H2 LXS51 68 M 26 2 1523516015 D 10 5 95 R2786H2 LXS52 61 F 27 2 1562224060 D 22 10 96 R2768H2 LXS52 61 M 27 2 1523516005 C 5 3 97 R2154H2 LXS54 70 F 27 2 1523516033 D 8 4 98 R2155H2 LXS54 70 M 27 2 1562224053 A 20 10 99 R1821H2 LXS55 77 F 25 2 1523516009 C 12 6 100 R1951H2 LXS55 74 M 26 2 1523516024 F 13 7 101 R2789H2 LXS56 71 F 25 2 1523516005 F 5 3 102 R2788H2 LXS56 71 M 25 2 1562224060 F 22 10 103 R2787H2 LXS59 66 F 29 2 1562224060 E 22 10 104 R2785H2 LXS59 62 M 29 2 1523516005 E 5 3 105 R1791H2 LXS60 58 F 27 2 1562224038 D 25 11 106 R1792H2 LXS60 64 M 27 2 1523516007 E 11 6 107 R1796H2 LXS62 58 F 27 2 1523516007 F 11 6 108 R1797H2 LXS62 58 M 27 2 1562224038 E 25 11 109 R2220H2 LXS64 71 F 28 2 1523516001 F 9 5 110 R2221H2 LXS64 71 M 28 2 1562224054 B 21 10 111 R1989H2 LXS66 73 F 26 2 1562224036 B 17 9 112 R1843H2 LXS66 78 M 27 2 1523516009 E 12 6 113 R2820H2 LXS68 67 F 29 2 1523516003 E 1 1 114 R2819H2 LXS68 67 M 29 2 1562224034 E 23 11 115 R1963H2 LXS7 78 F 28 2 1562224032 B 15 8 116 R1964H2 LXS7 78 M 28 2 1562224032 C 15 8 117 R2166H2 LXS70 72 F 27 2 1523516033 F 8 4 118 R2745H2 LXS70 71 M 28 2 1562224054 F 21 10 119 R2848H2 LXS72 72 F 27 2 1562224038 A 25 11 120 R1902H2 LXS72 66 M 27 2 1523516024 A 13 7 121 R2750H2 LXS73 81 F 25 2 1523516030 D 4 2 122 R1835H2 LXS73 90 M 24 2 1523516009 D 12 6 123 R1979H2 LXS75 59 F 27 2 1562224047 D 16 8 124 R2826H2 LXS75 72 M 27 2 1523516003 F 1 1 125 R2142H2 LXS76 77 F 26 2 1562224050 E 19 9 126 R1884H2 LXS76 85 M 26 2 1562224041 A 26 12 127 R1959H2 LXS78 69 F 26 2 1523516029 E 14 7 128 R1958H2 LXS78 69 M 26 2 1523516029 D 14 7 129 R2845H1 LXS8 70 F 28 2 1523516006 C 6 3 130 R2156H2 LXS8 76 M 27 2 1562224053 B 20 10 131 R1955H2 LXS80 71 F 25 2 1523516029 A 14 7 132 R1956H2 LXS80 71 M 25 2 1523516029 B 14 7 133 R2830H1 LXS84 66 F 26 2 1523516011 D 2 1 134 R2829H2 LXS84 66 M 26 2 1562224037 A 24 11 135 R2839H2 LXS86 68 F 27 2 1562224037 C 24 11 136 R2838H1 LXS86 68 M 27 2 1523516006 B 6 3 137 R2882H1 LXS87 66 F 27 2 1523516006 F 6 3 138 R2744H2 LXS87 71 M 26 2 1562224054 E 21 10 139 R2831H2 LXS88 69 F 27 2 1562224037 B 24 11 140 R2762H2 LXS88 71 M 27 2 1523516005 A 5 3 141 R2828H1 LXS89 75 F 26 2 1523516011 C 2 1 142 R1962H2 LXS89 73 M 25 2 1562224032 A 15 8 143 R1746H2 LXS9 66 F 26 2 1523516015 F 10 5 144 R2801H2 LXS9 68 M 27 2 1523516003 D 1 1 145 R1812H2 LXS90 61 F 25 2 1562224038 F 25 11 146 R1813H2 LXS90 61 M 25 2 1523516009 A 12 6 147 R1736H2 LXS92 66 F 23 2 1523516015 E 10 5 148 R1609H2 LXS92 87 M 23 2 1523516032 A 7 4 149 R1624H2 LXS93 74 F 26 2 1523516032 B 7 4 150 R1815H2 LXS93 61 M 26 2 1523516009 B 12 6 151 R1991H2 LXS94 70 F 25 2 1562224036 C 17 9 152 R2002H2 LXS94 70 M 25 2 1562224049 B 18 9 153 R1996H2 LXS96 75 F 23 2 1562224036 F 17 9 154 R1772H2 LXS96 63 M 23 2 1523516007 C 11 6 155 R2759H2 LXS97 73 F 26 2 1562224060 B 22 10 156 R2739H2 LXS97 79 M 26 2 1523516028 F 3 2 157 R2149H2 LXS98 78 F 26 2 1523516033 C 8 4 158 R1888H2 LXS98 76 M 26 2 1562224041 B 26 12 159 R1644H2 LXS99 79 F 26 2 1523516015 B 10 5 160 R2145H2 LXS99 77 M 27 2 1562224050 F 19 9

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. · 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.