From d029d5d7f8ead1f1de8d318045004a4a6f68f5fb Mon Sep 17 00:00:00 2001 From: Bonface Date: Fri, 9 Feb 2024 09:41:28 -0600 Subject: Update dataset RTF Files. --- general/datasets/EYE_M2_1105_P/tissue.rtf | 1554 +++++++++++++++++++++++++++++ 1 file changed, 1554 insertions(+) create mode 100644 general/datasets/EYE_M2_1105_P/tissue.rtf (limited to 'general/datasets/EYE_M2_1105_P/tissue.rtf') diff --git a/general/datasets/EYE_M2_1105_P/tissue.rtf b/general/datasets/EYE_M2_1105_P/tissue.rtf new file mode 100644 index 0000000..ec39390 --- /dev/null +++ b/general/datasets/EYE_M2_1105_P/tissue.rtf @@ -0,0 +1,1554 @@ +
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Tissue preparation protocol. Animal were killed by rapid cervical dislocation. Eyes were removed immediately and placed in RNAlater at room temperature. Usually six eyes from animals with a common sex, age, and strain were stored in a single tube.

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Each array was hybridized with a pool of RNA from 4 to 8 eyes from 2 to 4 animals. RNA was extracted at UTHSC by Zhiping Jia. If tissue was saved for RNA extraction at a later time, eyes were placed directly in RNAlater (Ambion, Inc.) and treated per the manufacturer’s directions. If eyes were used for immediate RNA extraction then we proceeded immediately to the next steps.

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Dissecting and preparing eyes for RNA extraction

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  1. Place eyes for RNA extraction in RNA STAT-60 (Tel-Test Inc.) and process per manufacturer’s instructions (in brief form below).
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  2. Store RNA in 75% ethanol at –80 deg. C until use.
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Total RNA was extracted with RNA STAT-60 (Tel-Test Inc.) according to the manufacturer's instructions. Briefly we:

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  1. homogenize tissue samples in the RNA STAT-60 (1 ml/50 to 100 mg tissue)
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  2. allowed the homogenate to stand for 5 min at room temperature
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  3. added 0.2 ml of chloroform per 1 ml RNA STAT-60
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  4. shook the sample vigorously for 15 sec and let the sample sit at room temperature for 3 min
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  5. centrifuged at 12,000 G for 15 min
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  6. transfered the aqueous phase to a fresh tube
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  7. added 0.5 ml of isopropanol per 1 ml RNA STAT-60
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  8. vortexed and allowed sample to stand at room temperature for 5-10 min
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  9. centrifuged at 12,000 G for 10-15 min
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  10. removed the supernatant and washed the RNA pellet with 75% ethanol
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  11. stored the pellet in 75% ethanol at -80 deg C until use
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Sample Processing. Samples were processed in the VA Medical Center, Memphis, Rheumatology Disease Research Core Center lead by Drs. John Stuart and Weikuan Gu. All processing steps were performed by Dr. Yan Jiao. In brief, samples were purified using a standard sodium acetate in alcohol method (recommended by Affymetrix). The RNA quality was checked using a 1% agarose gel. The 18S and 28S bands had to be clear and the 28S band had to be more prominent. RNA concentation was measured using a spectrophotometer. The 260/280 ratios had to be greater than 1.7, and the majority were 1.8 or higher. We used a total of 8 micrograms of RNA as starting amount for cDNA synthesis using a standard Eberwine T7 polymerase method (Superscript II RT, Invitrogen Inc., Affy Part No 900431, GeneChip Expression 3' Amplification One-Cyle cDNA Synthesis Kit). The Affymetrix IVT labeling kit (Affy 900449) was used to generate labeled cRNA. At this point the cRNA was evaluated again using both the 260/280 ratio (values of 2.0 or above were acceptable) and 1% agarose gel inspection of the product (a size range from 200 to 7000 bp is considered suitable for use). We used 45 micrograms of labeled cRNA for fragmentation. Those samples that passed both QC steps (<10% usually fail) were then sheared using a fragmentation buffer included in the Affymetrix GeneChip Sample Cleanup Module (Part No.900371). After fragmentation, samples were either stored at -80 deg. centrigrade until use (one third roughly) or were immediately used for hybridization.

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Dealing with Ocular Pigmentation: Variable ocular pigmentation is a potential confound in a study of the whole eye transcriptome. Even the most careful RNA preparations of brown and beige colored mice tend to have faint residual pigmentation that does affect hybridization signal. The key determinant of this interesting effect is the Tyrp1 (brown) locus on Chr 5 at about 80 Mb. Loci on Chr 4 that map at this location should be considered with skepticism and reviewed carefully. To address this problem Yan Jiao purified total RNA a second time using the Qiagen RNeasy MinElute Cleanup Kit (Cat No. 74204). This was done for 8 colored samples (R2534E2, R2578E2, R1441E2, R2537E2, R2536E2, R2589E2, R2539E2), of which 5 were finally included in this data set (cases in which the strain ID is labeled with asterisks in the table).

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Replication and Sample Balance: Our goal is to obtain data for independent biological sample pools from at least one of sample from each sex for all BXD strains. The current first batch of array data, represents a balanced sample of males and females, but without within-strain replication. We expect to add roughly 100 additional samples inthe next few months.

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Batch Structure: This data set consists of a single batch. The great majority of arrays are from a single lot.

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The table below summarizes information on strain, age, sex, original CEL filename, several quality control values, and source of mice. Columns labeled "PDNN 2Z outlier" and "RMA 2Z outlier" list the fraction of probe sets with values that deviated more than 2 z units from the mean. Scale factor, background average, present, absent, marginal and 3'/5' ratios for actin and Gapdh were collated from the Affymetrix Report (RPT) files.

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Idtube IDgroup typeStrainagesexoriginal CEL filenamePDNN 2Z outlierRMA 2Z outlierscale factorbackground averagepresentabsentmarginalAFFX-b-ActinMur(3'/5')AFFX-GapdhMur(3'/5')source
1R2607E1GDP BXDC57BL/6J67FR2605E.CEL0.0050.0092.428115.120.5860.40.0141.310.76UTM RW
2R0872E2GDP BXDC57BL/6J66MR0872E.CEL0.0130.0123.12888.580.5890.3960.0151.30.79UTM RW
3R2572E1GDP BXDDBA/2J65MR2572E.CEL0.0410.0512.40679.070.5550.4290.0161.370.79UTM RW
4R2601E1GDP BXDB6D2F173FR2601E.CEL0.0030.0042.54591.960.5890.3960.0151.440.78UTM RW
5R2602E1GDP BXDB6D2F173MR2602E.CEL0.0010.0042.59984.440.5970.3880.0151.370.78UTM RW
6R2600E1GDP BXDD2B6F172FR2600E.CEL0.0030.0082.4794.750.5810.4020.0171.410.78UTM RW
7R2604E1GDP BXDD2B6F169MR2604E.CEL0.0030.0072.65789.630.5940.3920.0151.280.79UTM RW
8R2597E1BXDBXD261MR2597E.CEL0.0030.0072.37493.560.6030.3830.0151.340.77Glenn
9R2591E1BXDBXD560FR2591E.CEL0.0510.0091.7136.480.5850.40.0151.330.78Glenn
10R2570E1BXDBXD665FR2570E.CEL0.0020.0061.98786.730.5850.40.0151.460.76UTM RW
11R2538E1BXDBXD877FR2538E.CEL0.0370.0281.905101.980.6120.3730.0151.520.79UTM RW
12R2569E1BXDBXD967MR2569E.CEL0.0140.0271.75387.360.5510.4340.0152.823.14UTM RW
13R2581E1BXDBXD1165FR2581E.CEL0.0060.0121.94188.550.6210.3640.0161.550.81UTM RW
14R2543E1BXDBXD1263MR2543E.CEL0.0360.0071.605117.690.5860.3990.0161.430.77UTM RW
15R2586E1BXDBXD1360FR2586E.CEL0.0200.0352.00673.610.5640.420.0162.853.81Glenn
16R2557E1BXDBXD1460FR2557E.CEL0.0140.0171.8398.760.6250.3610.0141.310.78Glenn
17R2567E1BXDBXD1660MR2567E.CEL0.0160.0252.23982.350.5670.4160.0171.370.75Glenn
18R2559E1BXDBXD1859MR2559E.CEL0.0350.0061.654103.680.6080.3770.0151.270.78Glenn
19R2560E1BXDBXD1960FR2560E.CEL0.0260.0071.79298.330.6090.3750.0161.350.8Glenn
20R2584E1BXDBXD2059FR2584E.CEL0.0030.0072.0783.820.5930.3910.0161.40.76Glenn
21R2541E2BXDBXD2161MR2541E2.CEL0.0490.0362.625125.080.560.4240.0151.290.78UTM RW
22R2553E1BXDBXD2258FR2553E.CEL0.0030.0051.952111.30.5990.3850.0151.280.76Glenn
23R2558E1BXDBXD2360FR2558E2.CEL0.0130.0151.908114.530.5990.3880.0141.20.82Glenn
24R2589E2BXDBXD24-rd*59MR2589E2.CEL0.0980.0982.606112.190.5750.4090.0161.240.8Glenn
25R2573E1BXDBXD2567FR2573E2.CEL0.0090.0183.15371.880.5790.4070.0141.770.97UAB
26R2562E1BXDBXD2860FR2562E.CEL0.0030.0051.649116.350.5990.3840.0171.370.79Glenn
27R2561E1BXDBXD2960FR2561E.CEL0.0190.0291.95293.320.5830.4020.0152.191Glenn
28R2598E1BXDBXD3161MR2598E.CEL0.0030.0061.989106.480.6090.3760.0151.270.78UTM RW
29R2563E1BXDBXD3263FR2563E.CEL0.0080.0111.547101.520.6190.3670.0141.50.8UTM RW
30R2542E1BXDBXD3367FR2542E.CEL0.0100.0162.12897.080.5650.4180.0161.910.93UTM RW
31R2585E1BXDBXD3460MR2585E.CEL0.0070.0142.6475.130.5830.40.0171.250.77Glenn
32R2532E1BXDBXD3862MR2532E.CEL0.0020.0032.03893.650.5980.3870.0151.370.8UTM RW
33R2574E1BXDBXD3970FR2574E.CEL0.0010.0041.98190.640.6120.3730.0151.390.78UTM RW
34R2590E1BXDBXD4060MR2590E.CEL0.0040.0072.70877.30.5910.3930.0151.40.77Glenn
35R2596E1BXDBXD4259MR2596E.CEL0.0130.0172.632108.460.590.3960.0151.240.8Glenn
36R2605E1BXDBXD4379MR2607E.CEL0.0030.0061.817131.220.6050.3820.0131.320.8UTM RW
37R2594E1BXDBXD4463FR2594E.CEL0.0040.0091.766117.330.5980.3880.0141.350.85UTM RW
38R2592E1BXDBXD4562MR2592E.CEL0.0020.0041.85106.160.6010.3860.0131.430.85UTM RW
39R2606E1BXDBXD4878MR2606E.CEL0.0030.0102.556106.160.5890.3970.0141.350.83UTM RW
40R2603E1BXDBXD5166FR2603E.CEL0.0030.0092.488115.160.5770.4080.0151.240.79UTM RW
41R2534E2BXDBXD61*70FR2534E2.CEL0.0300.0282.473117.760.5790.4060.0151.420.79UTM RW
42R2611E1BXDBXD6468MR2611E.CEL0.0130.0222.29291.990.580.4050.0151.571.06UTM RW
43R2583E1BXDBXD6560MR2583E.CEL0.0050.0102.49270.430.5690.4150.0161.671.01UTM RW
44R2536E2BXDBXD66*64FR2536E2.CEL0.0390.0652.74108.620.5610.4230.0171.280.79UTM RW
45R2551E1BXDBXD6867FR2551E.CEL0.0370.0392.49392.380.5430.4410.0162.911.55UTM RW
46R2593E1BXDBXD6959FR2593E.CEL0.0080.0131.672127.60.5920.3950.0131.470.92UTM RW
47R2537E2BXDBXD70*59MR2537E2.CEL0.0460.0442.9398.660.580.4050.0161.290.75UTM RW
48R2565E1BXDBXD7561FR2565E.CEL0.0090.0171.79101.680.580.4050.0152.313.47UTM RW
49R2579E1BXDBXD8065FR2579E.CEL0.0050.0102.41972.130.5920.3940.0151.730.82UTM RW
50R2540E1BXDBXD8763MR2540E.CEL0.0130.0162.33393.150.6110.3740.0141.220.81UTM RW
51R2545E1BXDBXD8967MR2546E.CEL0.0460.0461.667104.760.5620.4230.0153.69.84UTM RW
52R2578E2BXDBXD90*61FR2578E2.CEL0.0330.0342.78592.270.5860.3980.0161.520.77UTM RW
53R2554E1BXDBXD9667MR2554E.CEL0.0040.0042.17793.020.6020.3830.0151.460.77UTM RW
54R2577E1BXDBXD9755MR2577E.CEL0.0190.0162.0776.580.5950.3910.0141.871.29UTM RW
55R2595E1GDP129S1/SvImJ59FR2595E.CEL0.0170.0211.792115.390.610.3750.0151.460.77UTM RW
56R2533E1GDP129S1/SvImJ60MR2533E.CEL0.0210.0132.10793.550.5790.4050.0161.370.78UTM RW
57R2546E1GDPA/J66FR2545E.CEL0.0180.0141.98995.590.5860.3970.0171.470.78UTM RW
58R0754E2GDPA/J60MR0754E.CEL0.0140.0162.71885.630.5980.3870.0151.360.76JAX
59R1676E1GDPBALB/cByJ83FR1676E.CEL0.0420.0412.68598.370.5890.3960.0151.460.74JAX
60R1672E1GDPBALB/cByJ83MR1672E.CEL0.0220.0222.216110.520.5990.3860.0151.260.8JAX
61R1700E1GDPC3H/HeJ83FR1700E.CEL0.0900.0922.97868.770.6080.3790.0141.480.78UTM RW
62R1704E1GDPC3H/HeJ83MR1704E.CEL0.0860.0892.58188.290.6010.3860.0131.380.84UTM RW
63R2564E1GDPCAST/Ei64FR2564E.CEL0.0780.0641.93788.890.5850.3990.0161.60.77JAX
64R2580E1GDPCAST/Ei64MR2580E.CEL0.0760.0672.08994.640.5820.4010.0171.40.76JAX
65R2636E1GDPKK/HIJ64FR2636E.CEL0.0230.0262.6193.10.5890.3950.0151.390.76UTM RW
66R2637E1GDPKK/HIJ64MR2637E.CEL0.0390.0202.189102.780.5940.390.0151.30.79UTM RW
67R0999E1GDPLG/J57FR0999E.CEL0.0120.0122.44882.090.5940.3910.0151.380.79UTM RW
68R1004E1GDPLG/J65MR1004E.CEL0.0130.0152.43891.710.5870.3980.0151.380.79UTM RW
69R1688E1GDPNOD/LtJ66FR1688E.CEL0.0170.0192.66497.650.5860.3990.0151.260.8JAX
70R2566E1GDPNOD/LtJ76MR2566E2.CEL0.0190.0253.03169.440.5980.3880.0151.380.75UTM RW
71R2550E1GDPNZO/HlLtJ96MR2550E.CEL0.0230.0151.79487.160.6070.3780.0151.520.82JAX
72R2535E1GDPNZO/HlLtJ62FR2535E.CEL0.0460.0251.89385.670.6040.3820.0141.410.85JAX
73R2634E1GDPPWD/PhJ62FR2635E.CEL0.0770.0693.29289.80.5590.4250.0161.570.81JAX
74R2635E1GDPPWD/PhJ62MR2634E.CEL0.0880.0813.72280.050.5420.4410.0171.530.85JAX
75R2544E1GDPPWK/PhJ63FR2544E.CEL0.1060.1002.196107.510.5490.4350.0171.360.82JAX
76R2549E1GDPPWK/PhJ83MR2549E.CEL0.0650.0482.27583.80.5730.4120.0151.570.83JAX
77R2368E1GDPWSB/EiJ67FR2368E.CEL0.0250.0282.56785.70.5950.3910.0141.290.74UTM RW
78R2547E1GDPWSB/EiJ67MR2547E.CEL0.0320.0212.13590.040.5820.4010.0161.320.77UTM RW
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