HZI Treg CD4+CD25+ M430v2 (Nov06) RMA

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- - -This is the first regulatory T cell (CD4+CD25+) data set generated by Prof. Dr. -Klaus Schughart and colleagues at the Helmholtz Centre for Infection Research. - -Samples were processed using a total of 35 Affymetrix -Mouse Expression 430 2.0 short oligomer microarrays (MOE430 2.0), of which 33 passed stringent -quality control and error checking.

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This is the first data freeze and the set -is still private. Please contact Dr. Klaus Schughart for access.

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About the material used to generate this set of data:

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- -BXD spleen sample pools (from 2-3 mice) were obtained from a pathogen-free mice of the Dutch Mouse Phenomics Consortium (MPC) in Amsterdam. The mice were imported into the central animal -facility at the HZI and kept at a pathogen-free status. The mice were -euthanized using CO2 and spleenocytes prepared. At the -age of preparation, most mice were between 17 and 22 weeks of age.

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- -FACS sorting was applied to select the -CD4 positive T-cells. These cells were further separated into CD4+CD25+ and -CD4+CD25- pools.

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- -The BXD genetic reference population of recombinant inbred strains consists of approximately 80 strains. Approximately 800 classical phenotypes from sets of 10 to 70 of these strains been integrated -in the GeneNetwork. - -These data set includes expression values for 18 of the BXD strains made by Benjamin Taylor at the Jackson Laboratoryin 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.

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Affymetrix Mouse Genome 430 2.0 array: The 430v2 array consists -of 992,936 useful 25-nucleotide probes that estimate the expression of -approximately 39,000 transcripts and the majority of known genes and expressed -sequence tags. The array sequences were selected late in 2002 using Unigene Build 107 by Affymetrix. -The UTHSC group has recently reannotated all probe -sets on this array, producing more accurate data on probe and probe set -targets. All probes were aligned to the most recent assembly of the Mouse -Genome (Build 34, mm6) using Jim Kent's BLAT program. Many of the probe sets -have been manually curated by Jing -Gu and Rob Williams.

This data set was processed using the -RMA protocol. We then calculated the log base 2 of the intensity signal and -subsequently computed the Z scores for each value. We multiplied all Z scores -by 2 and added 8. The consequence of this simple set of transformations is to -produce a set of Z scores that have a mean of 8, a variance of 4, and a -standard deviation of 2. The advantage of this modified Z score is that a -two-fold difference in expression level (probe brightness level) corresponds -approximately to a 1 unit difference.

DataDesk was then used to examine the statistical quality of -the data. All except two fulfilled the stringency criteria, except for two arrays -BXD27f and BXD34m which were subsequently excluded from the set.

DataDesk allows a rapid detection of subsets of probes that -are particular sensitive to still unknown factors in array processing. Arrays -can then be categorized at the probe level into "reaction classes." A -reaction class is a group of arrays for which the expression of essentially all -probes are colinear over the full range of log2 -values. A single but large group of arrays (n = 32) processed in essentially -the identical manner by a single operator can produce arrays belonging to as -many as four different reaction classes. Reaction classes are NOT related to -strain, age, sex, treatment, or any known biological parameter (technical -replicates can belong to different reaction classes).

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 Klaus -Schughart and Dunja Bruder.