BXD animals were obtained from UTHSC, UAB, or directly from The Jackson Laboratory (see Table 1 below). Animals were housed at UTHSC, Beth Israel Deaconess, or the Jackson Laboratory before sacrifice. Virtually all CXB animals were obtained directly at the Jackson Laboratory by Lu Lu. We thank Muriel Davisson for making it possible to collect these cases on site. Standard inbred strain stock was from The Jackson Laboratory, but most animals were housed or reared at UTHSC. Mice were killed by cervical dislocation and brains were removed and placed in RNAlater prior to dissection. Cerebella and olfactory bulbs were removed; brains were hemisected, and both hippocampi were dissected whole by Hong Tao Zhang in the Lu lab. Hippocampal samples are very close to complete (see Lu et al., 2001) but probably include variable amounts of subiculum and fimbria.

A great majority of animals used in this study were between 45 and 90 days of age (average of 66 days, maximum range from 41 to 196 days; see Table 1 below). All animals were sacrifice between 9 AM and 5 PM during the light phase.

A pool of dissected tissue typically from six hippocampi and three naive adults of the same strain, sex, and age was collected in one session and used to generate cRNA samples. Two-hundred and one RNA samples were extracted at UTHSC by Zhiping Jia, four samples by Shuhua Qi (R2331H1, R2332H1, P2350H1, R2349H1), and one by Siming Shou (R0129H2).

RNA Extraction: In brief, we used the RNA STAT-60 protocol (TEL-TEST "B" Bulletin No. 1), steps 5.1A (homogenization of tissue), 5.2 (RNA extraction), 5.3 (RNA precipitation), and 5.4 (RNA wash). In Step 5.4 we stopped after adding 75% ethanol (1 ml per 1 ml RNA STAT-60) and stored the mix at -80°C until further use. Before RNA labeling we thawed samples and proceeded with the remainder of Step 5.4; pelleting, drying, and redissolving the pellet in RNase-free water.

We finally purify RNA by using Na4OAc before running arrays. Here is the detailed method:

Final RNA purification protocol

1. Add 1/10th volume of 3M Na4OAc , pH 5.2. If the sample was eluted with 100 µl nuclease-free water as suggested, this will be 10 µl of 3M Na4OAc.
2. Add 2.5 volumes of 100% ethanol (250 µl if the RNA was eluted in100 µl). Mix well and incubate at –20°C for 2 hrs.
3. Centrifuge at speed of 13,000 rpm for 20 min at 4°C. Carefully remove and discard the supernatant.
4. Wash the pellet with 800 µl 75% cold ethanol, centrifuge at speed of 8,600 rpm for 5 min, and remove the 75% ethanol. Wash again.
5. To remove the last traces of ethanol, quickly respin the tube, and aspirate any residual fluid.
6. Air dry the pellet.
7. Resuspend pellet in nuclease-free water.

5. PROTOCOL: RNA/mRNA isolation by the RNA STAT-60 method includes the following steps:
1. Homogenization RNA STAT-60TM (1 ml per 50-100 mg tissue, or 5-10 x 10-6 cells)
2. RNA Extraction 1 vol. of homogenate +0.2 vol. of chloroform
3. RNA Precipitation 0.5 vol. of isopropanol
4. RNA Wash 75% ethanol

Unless stated otherwise the procedure is carried out at room temperature.

5.1 HOMOGENIZATION

A. TISSUES: Homogenize tissues samples in the RNA STAT-60(1 ml/50-100mg tissue) in a glass-Teflon or Polytron homogenizer. Sample volume should not exceed 10% of the volume of the RNA STAT-60 used for homogenization.

B. CELLS: Cells grown in mono layer are lysed directly in a culture dish by adding the RNA STAT-60TM (1 ml/3.5 cm petri dish) and passing the cell lysate several times through a pipette. Cells grown in suspension are sediment then lysed in the RNA STAT-60TM (1 ml per 5-10 x 106 cells) by repetitive pipetting. Washing calls before addition of the RNA STAT-60TM should be avoided as this increases the possibility of mRNA degradation.

5.2 RNA EXTRACTION: Following homogenization, store the homogenate for 5 min at room temp to permit the complete dissociation of nucleoprotein complexes. Next, add 0.2 ml of chloroform per 1 ml of the RNA STAT-60, cover the sample tightly, shake vigorously for 15 seconds and let it stay at room temperature for 2-3minutes. Centrifuge the homogenate at 12,000g (max) for 15 minutes at 4°C. Following centrifugation, the homogenate separates into two phases: a lower red phenol chloroform phase and the colorless upper aqueous phase. RNA remains exclusively in the aqueous phase whereas DNA and proteins are in the interferes and organic phase. The volume of the aqueous phase is about 60% of the volume of RNA STAT-60 used for homogenization.

5.3 RNA PRECIPITATION: Transfer the aqueous phase to a fresh tube and mix with isopropanol. Add 0.5 ml of isopropanol per 1 ml of the RNA STAT-60 used for homogenization. Store samples at room temp for 5-10 minutes and centrifuge at 12,000g (max.) for 10 min at 4°C. RNA precipitate (often visible before centrifugation) forms a white pellet at the bottom of the tube.

5.4 RNA WASH: Remove supernatant and wash the RNA pellet once with 75% ethanol by vortexing and subsequent centrifugation at 7,500g (max.) for 5 min at 4°C. Add at least 1 ml of 75% ethanol per 1 ml of the RNA STAT-60 used for the initial homogenization.

At the end of the procedure, dry the RNA pellet briefly by air-drying or in a vacuum (5-10 min.). It is important not to let the RNA pellet dry completely as it will greatly decrease its solubility. Do not use the Speed-Vac for drying. Dissolve the RNA pellet in water or in 1 mm EDTA, pH 7, or 0.5% SDS solution. Vortex or pass the pellet a few times through a pipette tip. An incubation for 10-15 minutes at 55-60^oC may be required to dissolve RNA samples. Diethylpyrocarbonate (DEPC) treated RNase-free solutions¹ should be used for solubilization of RNA.

Sample Processing: Samples were processed in the INIA Bioanalytical Core at the W. Harry Feinstone Center for Genomic Research, The University of Memphis, led by Thomas R. Sutter. All processing steps were performed by Shirlean Goodwin. In brief, RNA purity was evaluated using the 260/280 nm absorbance ratio, and values had to be greater than 1.8. The majority of samples were 1.9 to 2.1. RNA integrity was assessed using the Agilent Bioanalyzer 2100. We required an RNA integrity number (RIN) of greater than 8. This RIN value is based on the intensity ratio and amplitude of 18S and 28S rRNA signals. The standard Eberwine T7 polymerase method was used to catalyze the synthesis of cDNA template from polyA-tailed RNA using Superscript II reverse transcriptase (Invitrogen Inc.). The Enzo Life Sciences, Inc., BioArray High Yield RNA Transcript Labeling Kit (T7, Part No. 42655) was used to synthesize labeled cRNA. The cRNA was evaluated using both the 260/280 ratio (values of 2.0 or 2.1 are acceptable) and the Bioanalyzer output (a dark cRNA smear on the 2100 output centered roughly between 600 and 2000 nucleotides is required). Those samples that passed both QC steps (10% usually failed and new RNA samples had to be acquired and processed) were then sheared using a fragmentation buffer included in the Affymetrix GeneChip Sample Cleanup Module (Part No. 900371). Fragmented cRNA samples were either stored at -80°C until use or were immediately injected onto the array. The arrays were hybridized and washed following standard Affymetrix protocols.

Replication and Sample Balance: Our goal was to obtain a male sample pool and female sample pool from each isogenic group. While almost all strains were orginally represented by matched male and female samples, not all data sets passed the final quality control steps. All but 5 of 99 strains (BXD55, BXD86, BXD94, BALB/cByJ, and CAST/EiJ) are represented by pairs or (rarely) trios of arrays. The first and last samples are technical replicates of a B6D2F1 hippocampal pool (aliquots R1291H3 and R1291H4).

Sex Balance: Based on the expression of Xist, probe set 1427262_at, DBA/2J and KK/HlJ are represented only by female samples, BXD55, and BALB/cByJ are only represented by a single male sample, BXD74 is represented by two male samples, and BXD86, BXD94, and CAST/EiJ are possibly mixed sex samples. One of the BXD9 samples, array R1523, may be a mixed sex sample pool because the expression of Xist is intermediate.

Experimental Design and Batch Structure: This data set consists arrays processed in six groups over a three month period (May 2005 to August 2005). Each group consists of 32 to 34 arrays. Sex, strain, and strain type (BXD, CXB, and MDP) were interleaved among groups to ensure reasonable balance and to minimize group-by-strain statistical confounds in group normalization. The two independent samples from a single strain were always run in different groups. All arrays were processed using a single protocol by a single operator, Shirlean Goodwin.

All samples in a group were labeled on one day, except for a few cases that failed QC on their first pass. The hybridization station accommodates up to 20 samples, and for this reason each group was split into a large first set of 20 samples and a second set of 12 to 14 samples. Samples were washed in groups of four and then held in at 4°C until all 20 (or 12-14) arrays were ready to scan. The last four samples out of the wash stations were scanned directly. Samples were scanned in sets of four.

COMPARISON with December 2005 Data Set: Both BXD14 arrays in the Dec05 data set were found to actually be from BXD23 cases. This error of strain identification has been corrected in the present data set. Four arrays in the Dec05 data set have been deleted because we judged them to be of poor quality (strain_sex_sample_firstreaction_group):

1. BXD21_F_1_1_G1
2. BXD23_M_1_1_G7
3. BXD36_M_1_1_G2
4. BXD36_F_1_1_G3

In the Dec05 data set there are a total of 1986 transcripts with QTLs that have LRS scores above 50, whereas in the corrected June06 data sets there are a total of 2074 transcripts with QTLs above 50.

Data Table 1:

This table lists all arrays by file order (Index), tube/sample ID, age, sex, batch, and numbers of animals in each sample pool (pool size). The next columns (RMA outlier, scale factor, background average, present, absent, marginal, AFFY-b-ActinMur(3'/5'), AFFY-GapdhMur(3'/5')) are all Affymetrix QC data. Finally, source lists the source colony of the animals. (Final version, fully corrected, by Arthur Centeno, October 2008)