HZI Treg M430v2 (Feb11) RMA

Summary

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.

Experiment design

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 strains DBA/2J and C57BL/6J, were isolated by flushing the spleens with erythrocyte lysis buffer. Cells were collected by centrifugation, resuspended 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 resuspended 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).

About cases

Parental and 31 BXD lines were studied. Mice were received from The 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 were 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).

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.

    Table 1

Index ProbeSet ID Sample Description Sex Strain cd25 Microarray Short Description Age Pool No. Pool members (animal number) Date of preparation 1 HZI1008 BXD-06f (f1) CD25+ F BXD6 CD25+ Yes BXD-06f 17 f1 1,3,4 1-31-2006 2 HZI1009 BXD-06m (m2) CD25+ M BXD6 CD25+ Yes BXD-06m 18 m2 5,6,7 1-31-2006 3 HZI1010 BXD-14m (m3) CD25+ M BXD14 CD25+ Yes BXD-14m 17 m3 1,3,4 1-31-2006 4 HZI1013 BXD-40f (f6) CD25+ F BXD40 CD25+ Yes BXD-40f 17 f6 1,2,3 2-1-2006 5 HZI1014 BXD-40m (m7) CD25+ M BXD40 CD25+ Yes BXD-40m 17 m7 5,6,7 2-2-2006 6 HZI1015 BXD-02f (f8) CD25+ F BXD2 CD25+ Yes BXD-02f 17 f8 1,2,3 2-14-2006 7 HZI1016 BXD-02m (m20) CD25+ M BXD2 CD25+ Yes BXD-02m 21 m20 4,5,6 4-6-2006 8 HZI1017 BXD-11f (f30) CD25+ F BXD11 CD25+ Yes BXD-11f 17 f30 3,4,5 5-11-2006 9 HZI1018 BXD-11m (m9) CD25+ M BXD11 CD25+ Yes BXD-11m 18 m9 1,2 2-14-2006 10 HZI1019 BXD-12f (f10) CD25+ F BXD12 CD25+ Yes BXD-12f 17 f10 1,2,3 2-14-2006 11 HZI1020 BXD-39f (f23) CD25+ F BXD39 CD25+ Yes BXD-39f 19 f23 4,5,6 4-11-2006 12 HZI1021 BXD-33m (m11) CD25+ M BXD33 CD25+ Yes BXD-33m 17 m11 1,2 2-14-2006 13 HZI1022 BXD-18f (f14) CD25+ F BXD18 CD25+ Yes BXD-18f 17 f14 3,4,5 2-15-2006 14 HZI1023 BXD-18m (m13) CD25+ M BXD18 CD25+ Yes BXD-18m 18 m13 7,8 2-15-2006 15 HZI1024 BXD-23m (m15) CD25+ M BXD23 CD25+ Yes BXD-23m 18 m15 1,2,3 2-15-2006 16 HZI1026 BXD-09f (f17) CD25+ F BXD9 CD25+ Yes BXD-09f 21 f17 1,2,3 4-5-2006 17 HZI1028 BXD-09m (m35) CD25+ M BXD9 CD25+ Yes BXD-09m 15 m35 7,8,9 7-7-2006 18 HZI1029 BXD-32f (f18) CD25+ F BXD32 CD25+ Yes BXD-32f 21 f18 1,2,3 4-6-2006 19 HZI1030 BXD-32m (m19) CD25+ M BXD32 CD25+ Yes BXD-32m 22 m19 1,2,3 4-6-2006 20 HZI1031 BXD-33f (f22) CD25+ F BXD33 CD25+ Yes BXD-33f 18 f22 2,3,4 4-11-2006 21 HZI1032 BXD-39m (m29) CD25+ M BXD39 CD25+ Yes BXD-39m 17 m29 5,6,7 5-10-2006 22 HZI1033 BXD-01f (f32) CD25+ F BXD1 CD25+ Yes BXD-01f 18 f32 3,4 7-6-2006 23 HZI1034 BXD-01m (m31) CD25+ M BXD1 CD25+ Yes BXD-01m 18 m31 1,2 7-6-2006 24 HZI1035 BXD-16f (f26) CD25+ F BXD16 CD25+ Yes BXD-16f 18 f26 1,2,3 4-12-2006 25 HZI1036 BXD-21f (f25) CD25+ F BXD21 CD25+ Yes BXD-21f 19 f25 5,6,7 4-12-2006 26 HZI1037 BXD-21m (m24) CD25+ M BXD21 CD25+ Yes BXD-21m 18 m24 1,2,3 4-12-2006 27 HZI1039 BXD-31f (f34) CD25+ F BXD31 CD25+ Yes BXD-31f 16 f34 1,2,3 7-7-2006 28 HZI1040 C57BL/6Jf (f28) CD25+ F C57BL/6J CD25+ Yes C57BL/6Jf 16 f28 1,2,3 5-10-2006 29 HZI1041 DBA/2Jf (f27) CD25+ F DBA/2J CD25+ Yes DBA/2Jf 16 f27 5,6,7 5-10-2006 30 HZI1042 DBA/2Jm (m21) CD25+ M DBA/2J CD25+ Yes DBA/2Jm 21 m21 1,2,3 4-11-2006 31 HZI1487 BXD-08f (f67) CD25+ F BXD8 CD25+ Yes BXD-08f 11 f67 4,5,6 6-25-2007 32 HZI1488 BXD-08m (m66) CD25+ M BXD8 CD25+ Yes BXD-08m 17 m66 1,2,3 6-25-2007 33 HZI1489 BXD-16m (m36) CD25+ M BXD16 CD25+ Yes BXD-16m 20, 16 m36 5,6,7 8-28-2006 34 HZI1490 BXD-12m (m42) CD25+ M BXD12 CD25+ Yes BXD-12m 20 m42 5,6,7 10-23-2006 35 HZI1491 BXD-13f (f44) CD25+ F BXD13 CD25+ Yes BXD-13f 15 f44 1,2,3 12-13-2006 36 HZI1492 BXD-13m (m45) CD25+ M BXD13 CD25+ Yes BXD-13m 15 m45 4,5,6,7 12-13-2006 37 HZI1493 BXD-14f (f48) CD25+ F BXD14 CD25+ Yes BXD-14f 16 f48 5,6,7 2-15-2007 38 HZI1494 BXD-19f (f64) CD25+ F BXD19 CD25+ Yes BXD-19f 19 f64 7,8,9 6-20-2007 39 HZI1495 BXD-19m (m46) CD25+ M BXD19 CD25+ Yes BXD-19m 16 m46 4,5,6 12-15-2006 40 HZI1499 BXD-28m (m43) CD25+ M BXD28 CD25+ Yes BXD-28m 17,2 m43 1,2,3 10-23-2006 41 HZI1500 BXD-42f (f49) CD25+ F BXD42 CD25+ Yes BXD-42f 17 f49 ?? 3-8-2007 42 HZI1502 F1 (BXD)m (f50) CD25+ M B6D2F1 CD25+ Yes F1 (BXD)m 15 m51 1,2,3, 4-18-2007 43 HZI1503 F1 (BXD)m (m51) CD25+ F B6D2F1 CD25+ Yes F1 (BXD)f 15 f50 1,2,3 4-18-2007 44 HZI1504 BXD-86f (f52) CD25+ F BXD86 CD25+ Yes BXD-86f 16 f52 1,2,3 4-18-2007 45 HZI1505 BXD-43f (f53) CD25+ F BXD43 CD25+ Yes BXD-43f 16 f53 1,2,3 4-23-2007 46 HZI1506 BXD-44f (f54) CD25+ F BXD44 CD25+ Yes BXD-44f 18 f54 1,2,3 4-23-2007 47 HZI1507 BXD-45f (f55) CD25+ F BXD45 CD25+ Yes BXD-45f 19 f55 1,2,3 4-23-2007 48 HZI1508 BXD-62f (f56) CD25+ F BXD62 CD25+ Yes BXD-62f 17 f56 1,2,3 4-26-2007 49 HZI1509 BXD-73f (f57) CD25+ F BXD73 CD25+ Yes BXD-73f 18 f57 1,2,3 4-26-2007 50 HZI1510 BXD-51f (f59) CD25+ F BXD51 CD25+ Yes BXD-51f 22 f59 1,2,3 6-18-2007 51 HZI1523 BXD-75f (f58) CD25+ F BXD75 CD25+ Yes BXD-75f 15,17 f58 1,2,3 4-26-2007 52 HZI1525 BXD-29m (m37) CD25+ M BXD29 CD25+ Yes BXD-29m 20, 16 m37 1,2,3 8-29-2006 53 HZI1526 BXD-34f (f4) CD25+ F BXD34 CD25+ Yes BXD-34f 17 f4 1,2,3 2-1-2006 54 HZI1940 BXD-27m (m39) CD25+ M BXD27 CD25+ Yes BXD-27m 18 - 20 m39 1,3,4 9-1-2006 55 HZI1941 BXD-42m (m47) CD25+ M BXD42 CD25+ Yes BXD-42m 15,16 m47 1,2,3 12-15-2006 56 HZI1942 BXD-34m (m5) CD25+ M BXD34 CD25+ Yes BXD-34m 17 m5 5,7,8 2-1-2006 57 HZI1943 BXD-38f (f70) CD25+ F BXD38 CD25+ Yes BXD-38f 13 f70 4,5,6,7 2-1-2008 58 HZI1944 BXD-31m (m69) CD25+ M BXD31 CD25+ Yes BXD-31m 14 m69 4,5,6 2-1-2008 59 HZI1945 BXD-27f (f12) CD25+ F BXD27 CD25+ Yes BXD-27f 18 f12 1,2 2-15-2006 60 HZI1946 BXD-38m (m63) CD25+ M BXD38 CD25+ Yes BXD-38m 18 m63 1,2,3 6-20-2007 61 HZI1947 BXD-23f (f62) CD25+ F BXD23 CD25+ Yes BXD-23f 21 f62 1,2,3 6-20-2007 62 HZI1948 BXD-28f (f61) CD25+ F BXD28 CD25+ Yes BXD-28f 22 f61 1,2,3 6-18-2007

About platform

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.

About data processing

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.
y_i = μ + B_i + e_i
Where y_i is the expression level of the i^th microarray, μ is the overall mean, B_i is the batch to which the ith individual belongs and e_i 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).

Acknowledgment

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.

Notes

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