From d0911a04958a04042da02a334ccc528dae79cc17 Mon Sep 17 00:00:00 2001 From: zsloan Date: Fri, 27 Mar 2015 20:28:51 +0000 Subject: Removed everything from 'web' directory except genofiles and renamed the directory to 'genotype_files' --- web/dbdoc/RTC_1106_R.html | 236 ---------------------------------------------- 1 file changed, 236 deletions(-) delete mode 100755 web/dbdoc/RTC_1106_R.html (limited to 'web/dbdoc/RTC_1106_R.html') diff --git a/web/dbdoc/RTC_1106_R.html b/web/dbdoc/RTC_1106_R.html deleted file mode 100755 index eae25179..00000000 --- a/web/dbdoc/RTC_1106_R.html +++ /dev/null @@ -1,236 +0,0 @@ - -About the HZI Regulatory T Cell mRNA data set of Feb 2011 on GN - - - - - - - - - - - - - - - - - -
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- -Helmholtz Zentrum für Infektionsforschung (HZI) T-Regulatory Cell Affymetrix M430v2 February 2011 RMA Data Set modify this page

Accession number: GN122

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    Summary:

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-ERROR-CHECKED FIRST PHASE PRIVATE TEST DATA SET. This data set provides estimates of gene expression in regulatory T cells (CD4+CD25+) of BXD strains. Data were generated by Prof. Dr. Klaus Schughart and colleagues at the Helmholtz Centre for Infection Research (HZI). Samples were processed using a total of 35 Affymetrix MOE 430 2.0 short oligomer microarrays, of which 33 passed stringent quality control and error checking. - -

This is a private test data set. Please contact Dr. Klaus Schughart for early access. - - -

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    About the cases used to generate this set of data:

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-Parental and 31 BXD lines were studied. Mice were received from Jackson Laboratory, or from The Oak Ridge National and were bred in the facility of the Neuro-BSIK consortium (VU University Amsterdam). The data set includes expression values for 18 of the BXD strains made by Benjamin Taylor at the Jackson Laboratory in the 1970s and 1990s (BXD1 through BXD40, as well as the two parental strains, C57BL/6J and DBA/2J. All of these strains are fully inbred, many well beyond the 100th filial (F) generation of inbreeding. - -

BXD spleen sample pools (from 2-3 mice) were obtained from a pathogen-free mice of the Dutch Mouse Phenomics Consortium (MPC) in Amsterdam. Mice were imported into the central animal -facility at the HZI and kept in a pathogen-free vivarium. Mice were euthanized using CO2 and spleenocytes wre prepared. Most mice were between 17 and 22 weeks of age when samples were collected. FACS sorting was used to select the CD4-positive T cells. These cells were further separated into CD4+CD25+ and CD4+CD25- pools. - -

Error-checking strain identity. A set of more than 20 probe sets with Mendelian segregation patterns in this HZI data set were used to confirm strain identify in early June, 2007. Two errors were detected and rectified. As of June 22, 2007, data are registered correctly. Prior to June 22, 2007, data listed as strains BXD33 and BXD39 were essentially hybrid (mixed) data sets. - -

On Aug 23, 2007, we loaded the final QTL Reaper data into GeneNetwork for the corrected data set. The maximum LRS generated by any probe set is 84.6 for 1436240_at (Tra2a). A total of 41 probe sets are associated with QTLs that have LRS values above 46 (LOD > 10). - -

Sex of samples is listed below in Table 1. In brief, data for BXD14 and 23 are male-only samples, whereas BXD12, 16, 31, 34, 36 and C57BL/6J are from female-only samples. All other samples (DBA/2J, BXD1, 2, 6, 9, 11, 18, 21 32, 33, 39, 40) consist of one male and one female array. The sex of samples can be independently validated using the Xist probe set (1427262_at). - - -

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Figure 1: The expression of Xist can be used as an independent marker for sex. Xist is expressed at very low levels (noise) in male samples (far left) and at high values in females (far right). Sex-balanced samples (middle) have high variance due to the inclusion of one array per sex.

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    Table 1

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IndexProbeSet IDSample DescriptionSexStraincd25MicroarrayShort DescriptionAgePool No.Pool members (animal number)Date of preparation
1HZI1008BXD-06f (f1) CD25+FBXD6CD25+YesBXD-06f17f11,3,41-31-2006
2HZI1009BXD-06m (m2) CD25+MBXD6CD25+YesBXD-06m18m25,6,71-31-2006
3HZI1010BXD-14m (m3) CD25+MBXD14CD25+YesBXD-14m17m31,3,41-31-2006
4HZI1013BXD-40f (f6) CD25+FBXD40CD25+YesBXD-40f17f61,2,32-1-2006
5HZI1014BXD-40m (m7) CD25+MBXD40CD25+YesBXD-40m17m75,6,72-2-2006
6HZI1015BXD-02f (f8) CD25+FBXD2CD25+YesBXD-02f17f81,2,32-14-2006
7HZI1016BXD-02m (m20) CD25+MBXD2CD25+YesBXD-02m21m204,5,64-6-2006
8HZI1017BXD-11f (f30) CD25+FBXD11CD25+YesBXD-11f17f303,4,55-11-2006
9HZI1018BXD-11m (m9) CD25+MBXD11CD25+YesBXD-11m18m91,22-14-2006
10HZI1019BXD-12f (f10) CD25+FBXD12CD25+YesBXD-12f17f101,2,32-14-2006
11HZI1020BXD-39f (f23) CD25+FBXD39CD25+YesBXD-39f19f234,5,64-11-2006
12HZI1021BXD-33m (m11) CD25+MBXD33CD25+YesBXD-33m17m111,22-14-2006
13HZI1022BXD-18f (f14) CD25+FBXD18CD25+YesBXD-18f17f143,4,52-15-2006
14HZI1023BXD-18m (m13) CD25+MBXD18CD25+YesBXD-18m18m137,82-15-2006
15HZI1024BXD-23m (m15) CD25+MBXD23CD25+YesBXD-23m18m151,2,32-15-2006
16HZI1026BXD-09f (f17) CD25+FBXD9CD25+YesBXD-09f21f171,2,34-5-2006
17HZI1028BXD-09m (m35) CD25+MBXD9CD25+YesBXD-09m15m357,8,97-7-2006
18HZI1029BXD-32f (f18) CD25+FBXD32CD25+YesBXD-32f21f181,2,34-6-2006
19HZI1030BXD-32m (m19) CD25+MBXD32CD25+YesBXD-32m22m191,2,34-6-2006
20HZI1031BXD-33f (f22) CD25+FBXD33CD25+YesBXD-33f18f222,3,44-11-2006
21HZI1032BXD-39m (m29) CD25+MBXD39CD25+YesBXD-39m17m295,6,75-10-2006
22HZI1033BXD-01f (f32) CD25+FBXD1CD25+YesBXD-01f18f323,47-6-2006
23HZI1034BXD-01m (m31) CD25+MBXD1CD25+YesBXD-01m18m311,27-6-2006
24HZI1035BXD-16f (f26) CD25+FBXD16CD25+YesBXD-16f18f261,2,34-12-2006
25HZI1036BXD-21f (f25) CD25+FBXD21CD25+YesBXD-21f19f255,6,74-12-2006
26HZI1037BXD-21m (m24) CD25+MBXD21CD25+YesBXD-21m18m241,2,34-12-2006
27HZI1039BXD-31f (f34) CD25+FBXD31CD25+YesBXD-31f16f341,2,37-7-2006
28HZI1040C57BL/6Jf (f28) CD25+FC57BL/6JCD25+YesC57BL/6Jf16f281,2,35-10-2006
29HZI1041DBA/2Jf (f27) CD25+FDBA/2JCD25+YesDBA/2Jf16f275,6,75-10-2006
30HZI1042DBA/2Jm (m21) CD25+MDBA/2JCD25+YesDBA/2Jm21m211,2,34-11-2006
31HZI1487BXD-08f (f67) CD25+FBXD8CD25+YesBXD-08f11f674,5,66-25-2007
32HZI1488BXD-08m (m66) CD25+MBXD8CD25+YesBXD-08m17m661,2,36-25-2007
33HZI1489BXD-16m (m36) CD25+MBXD16CD25+YesBXD-16m20, 16m365,6,78-28-2006
34HZI1490BXD-12m (m42) CD25+MBXD12CD25+YesBXD-12m20m425,6,710-23-2006
35HZI1491BXD-13f (f44) CD25+FBXD13CD25+YesBXD-13f15f441,2,312-13-2006
36HZI1492BXD-13m (m45) CD25+MBXD13CD25+YesBXD-13m15m454,5,6,712-13-2006
37HZI1493BXD-14f (f48) CD25+FBXD14CD25+YesBXD-14f16f485,6,72-15-2007
38HZI1494BXD-19f (f64) CD25+FBXD19CD25+YesBXD-19f19f647,8,96-20-2007
39HZI1495BXD-19m (m46) CD25+MBXD19CD25+YesBXD-19m16m464,5,612-15-2006
40HZI1499BXD-28m (m43) CD25+MBXD28CD25+YesBXD-28m17,2m431,2,310-23-2006
41HZI1500BXD-42f (f49) CD25+FBXD42CD25+YesBXD-42f17f49??3-8-2007
42HZI1502F1 (BXD)m (f50) CD25+MB6D2F1CD25+YesF1 (BXD)m15m511,2,3,4-18-2007
43HZI1503F1 (BXD)m (m51) CD25+FB6D2F1CD25+YesF1 (BXD)f15f501,2,34-18-2007
44HZI1504BXD-86f (f52) CD25+FBXD86CD25+YesBXD-86f16f521,2,34-18-2007
45HZI1505BXD-43f (f53) CD25+FBXD43CD25+YesBXD-43f16f531,2,34-23-2007
46HZI1506BXD-44f (f54) CD25+FBXD44CD25+YesBXD-44f18f541,2,34-23-2007
47HZI1507BXD-45f (f55) CD25+FBXD45CD25+YesBXD-45f19f551,2,34-23-2007
48HZI1508BXD-62f (f56) CD25+FBXD62CD25+YesBXD-62f17f561,2,34-26-2007
49HZI1509BXD-73f (f57) CD25+FBXD73CD25+YesBXD-73f18f571,2,34-26-2007
50HZI1510BXD-51f (f59) CD25+FBXD51CD25+YesBXD-51f22f591,2,36-18-2007
51HZI1523BXD-75f (f58) CD25+FBXD75CD25+YesBXD-75f15,17f581,2,34-26-2007
52HZI1525BXD-29m (m37) CD25+MBXD29CD25+YesBXD-29m20, 16m371,2,38-29-2006
53HZI1526BXD-34f (f4) CD25+FBXD34CD25+YesBXD-34f17f41,2,32-1-2006
54HZI1940BXD-27m (m39) CD25+MBXD27CD25+YesBXD-27m18 - 20m391,3,49-1-2006
55HZI1941BXD-42m (m47) CD25+MBXD42CD25+YesBXD-42m15,16m471,2,312-15-2006
56HZI1942BXD-34m (m5) CD25+MBXD34CD25+YesBXD-34m17m55,7,82-1-2006
57HZI1943BXD-38f (f70) CD25+FBXD38CD25+YesBXD-38f13f704,5,6,72-1-2008
58HZI1944BXD-31m (m69) CD25+MBXD31CD25+YesBXD-31m14m694,5,62-1-2008
59HZI1945BXD-27f (f12) CD25+FBXD27CD25+YesBXD-27f18f121,22-15-2006
60HZI1946BXD-38m (m63) CD25+MBXD38CD25+YesBXD-38m18m631,2,36-20-2007
61HZI1947BXD-23f (f62) CD25+FBXD23CD25+YesBXD-23f21f621,2,36-20-2007
62HZI1948BXD-28f (f61) CD25+FBXD28CD25+YesBXD-28f22f611,2,36-18-2007
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-About the Array Platform: -

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The Affymetrix M430 2.0 array consists of approximately 992,936 useful 25-nucleotide probes that estimate the expression of approximately 39,000 transcripts, including a majority of known genes and expressed sequence tags. The array sequences were selected late in 2002 using NCBI Build 107 by Affymetrix. The UTHSC GN group continuously reannotated probe sets on this array, producing more accurate data on probe and probe set targets. All probes have also be aligned to the most recent assembly of the Mouse Genome using Jim Kent's BLAT program. -

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Methods:

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Parental and BXD lines were received from Jackson Laboratory, or from Oak Ridge Laboratory (BXD43, BXD51, BXD61, BXD62, BXD65, BXD68, BXD69, BXD73, BXD75, BXD87, BXD90), and were bred in the facility of the Neuro-BSIK consortium (VU University Amsterdam). Female mice 3 per strain were housed on sawdust in standard Makrolon type II cages with food (Harlan Teklad 2018) and water ad libitum under specific pathogen free conditions. For the analysis, mice were transferred to the animal facility in Braunschweig and adapted for at least two weeks to the new environment before preparing the spleen cells. All protocols involving mice were approved by national animal welfare committees.

- For sorting of Tregs and Th cells, splenocytes from 31 BXD recombinant inbred strains as well as from the parental mouse lines DBA/2J and C57BL/6J were isolated by flushing the spleens with erythrocyte-lysis-buffer. Cells were collected by centrifugation, re-suspended in cold FACS-buffer (PBS / 2% FCS / 0,5 mM EDTA). After passing the cells through a 100 µm cell strainer and an additional washing step with FACS-buffer, splenocytes were stained with anti-CD4-APC and anti-CD25-PE for 10 minutes at 4°C, washed and re-suspended in FACS-buffer. CD4+ T cells were separated into CD4+CD25+ Tregs and CD4+CD25- Th cells using a MoFlo cell sorter (Cytomation) and purity of the sorted T cell subsets reached 95-97%.

- Quality and integrity of the total RNA isolated from 1x105 cells was controlled by running all samples on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). RNA amplification and labeling was done according to manufactures protocol (Small Sample Target Labeling Assay Version II, Affymetrix; Santa Clara, CA).  The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 10 µg of each biotinylated cRNA preparation were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix MOE430 2.0 for 16 hours at 46°C. After hybridisation the GeneChips were washed and stained using the Affymetrix´s recommended EukGE-WS2v5 protocol for GeneChip®  Fluidics FS400 station.  Images were scanned using GeneChip® Scanner 3000 under the control of GCOS 1.3 software package (Affymetrix; Santa Clara, CA).

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About the data processing: - -

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Microarray data then was preprocessed using the RMA method [bolstad] and subsequently batch corrected [Alberts et al]. In this study, RNA was extracted at three different points in time for the Treg samples and also microarray processing was performed at three different points in time. Similarly, the Th samples were processed in two batches. Therefore, we performed a batch correction for both cell types using the following ANOVA model before further analysis of the data.
-yi = μ + Bi + ei
-Where yi is the expression level of the ith microarray, μ is the overall mean, Bi is the batch to which the ith individual belongs and ei is the residual error.
-Batch corrected data sets were then preprocessed before transferring them to the GeneNetwork (GN) database: Adding an offset of 1 unit to each signal intensity value to ensure that the logarithm of all values were positive, computing the log2 value, performing a quantile normalization of the log2 values for the total set of arrays using the same initial steps used by the RMA transform, computing the Z scores for each cell value, multiplying all Z scores by 2 and adding 8 to the value of all Z scores. The advantage of this variant of a Z transformation is that all values are positive and that 1 unit represents approximately a 2-fold difference in expression as determined using the spike-in control probe sets. The mean values were subsequently calculated if multiple samples from one BXD line were recorded (male and females or replicates).

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Acknowledgment:

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These data were generated by Prof. Dr. Klaus Schughart (Department of Experimental Mouse Genetics) and Dr. Dunja Bruder (Research Group Immune Regulation) at the Helmholtz Center for Infection Research with the help of Dr. Lothar Gröbe (FACS sorting, Research Group Mucosal Immunity). - -

Funding was provided by the Helmholtz Association and publicly funded research projects awarded to Drs. Klaus -Schughart and Dunja Bruder.

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About this text file:

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This text file was generated by KS on July, 18 2011. -

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