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Summary
We profiled the liver transcriptome, proteome, and metabolome from 347 individuals from 58 isogenic strains of the BXD mouse population across age (7 to 24 months) and diet (low or high fat) to link molecular variations to metabolic traits. Several hundred genes are affected by diet and/or age at the transcript and protein levels. Orthologs of two aging-associated genes, St7 and Ctsd, were knocked down in C. elegans, reducing longevity in wildtype and mutant long-lived strains. The multiomics data were analyzed as segregating gene networks according to each independent variable, providing causal insight into segregating edges. Candidate hits were cross-examined in an independent Diversity Outbred mouse liver dataset segregating for similar diets, with ~80-90% of diet-related candidate genes found in common across datasets. Together, we have developed a large multiomics resource for multivariate analysis of complex traits and demonstrate a methodology for moving from observational associations to causal connections.
Experiment design
RNA sequencing
About cases
About the cases used to generate this set of data: All animal care was handled according to the NIH's Guidelines for the Care and Use of Laboratory Animals and was also approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center (UTHSC). 2157 mice from 89 strains of the BXD family (including parents and both F1s) were followed in the colony. 159 animals were males and 1998 animals were females. Animals were maintained in the UTHSC vivarium in Specific Pathogen-Free (SPF) housing throughout the longevity experiment. The housing environment was a 12-hour day/night cycle in 20–24°C temperature with housing cages of 145 in2 with up to 10 animals per cage. Diets were either Harlan Teklad 2018 (CD; 24% calories from protein, 18% from fat, 58% from carbohydrates) or Harlan Teklad 06414 (HFD; 18.3% calories from protein, 60.3% from fat, 21.4% from carbohydrates). Water was Memphis city municipal tap water. Food and water were ad libitum. All animals were followed from their point of entry into the colony (typically around 5 months of age) until death. Animals were checked daily for morbidity and were weighed approximately every 2-3 months throughout their lives. 662 animals were sacrificed at specific ages for tissue collection across cohort (i.e. diet, strain, sex, and age). These 662 animals were selected for tissue harvest with the following aims: 2 animals per strain, diet, and age, for a target of 4 age points, i.e. up to a target maximum of 16 sacrificed animals per strain (2 replicates * 2 diets * 4 ages). Transcriptomics were acquired for 291 individuals, which had targeted data generation for 1 animal of each of the 309 cohorts (i.e of the 662 individuals, just over half had a biological replicate which was age, diet, strain, and sex-matched). The target ages were 7, 12, 18, and 24 months of age. Roughly every 3 months for the duration of the experiment, ~40 animals were selected for sacrifice, with approximately 15 animals sacrificed per day over the course of 3 or 4 continuous days. Animals were removed from the aging colony the night prior to sacrifice, but retained access to food and water.
| CaseID | Strain | Diet | Sex | Age | EarTag current | GN-SampleID |
|---|---|---|---|---|---|---|
| 062013.09 | C57BL/6J | HF | F | 544 | 514 | H0514 |
| 091914.09 | C57BL/6J | CD | F | 537 | 1103 | H1103 |
| 012615.26 | C57BL/6J | CD | M | 361 | 1590 | H1590 |
| 102414.06 | C57BL/6J | HF | F | 182 | 1681 | H1681 |
| 062013.12 | D2B6F1 | CD | F | 545 | 646 | H0646 |
| 062013.13 | D2B6F1 | HF | F | 540 | 642 | H0642 |
| 101713.20 | D2B6F1 | HF | F | 214 | 1150 | H1150 |
| 101713.18 | D2B6F1 | CD | F | 210 | 1154 | H1154 |
| 083016.07 | DBA/2J | CD | F | 764 | 1818 | H1818 |
| 091914.05 | DBA/2J | HF | F | 549 | 1142 | H1142 |
| 091914.07 | DBA/2J | HF | M | 544 | 1140 | H1140 |
| 101713.10 | DBA/2J | CD | F | 212 | 1147 | H1147 |
| 101713.11 | DBA/2J | HF | F | 212 | 1144 | H1144 |
| 121214.29 | B6D2F1 | CD | F | 640 | 1227 | H1227 |
| 012615.07 | B6D2F1 | CD | F | 552 | 1569 | H1569 |
| 102616.04 | B6D2F1 | HF | F | 547 | 2288 | H2288 |
| 102616.05 | B6D2F1 | HF | F | 547 | 2290 | H2290 |
| 101713.13 | B6D2F1 | CD | F | 216 | 1223 | H1223 |
| 101713.15 | B6D2F1 | HF | F | 216 | 1302 | H1302 |
| 082214.11 | BXD9 | CD | F | 548 | 1006 | H1006 |
| 082214.09 | BXD9 | HF | F | 548 | 1009 | H1009 |
| 102616.12 | BXD9 | CD | F | 245 | 2577 | H2577 |
| 083016.08 | BXD24 | HF | F | 688 | 2259 | H2259 |
| 012615.19 | BXD24 | CD | F | 205 | 1792 | H1792 |
| 012615.08 | BXD29 | CD | F | 724 | 1044 | H1044 |
| 111314.10 | BXD29 | HF | F | 630 | 1037 | H1037 |
| 061913.04 | BXD29 | HF | F | 577 | 669 | H0669 |
| 111314.11 | BXD29 | CD | F | 574 | 1040 | H1040 |
| 061913.01 | BXD29 | CD | F | 572 | 742 | H0742 |
| 121515.05 | BXD29 | HF | F | 182 | 2349 | H2349 |
| 101014.02 | BXD32 | HF | F | 539 | 1137 | H1137 |
| 083016.01 | BXD32 | CD | F | 386 | 2494 | H2494 |
| 042015.22 | BXD32 | CD | F | 200 | 2034 | H2034 |
| 042015.20 | BXD32 | HF | F | 200 | 2030 | H2030 |
| 101513.05 | BXD34 | CD | F | 554 | 765 | H0765 |
| 101014.05 | BXD34 | HF | F | 537 | 1052 | H1052 |
| 101513.02 | BXD34 | CD | F | 201 | 1054 | H1054 |
| 101513.04 | BXD34 | HF | F | 197 | 1056 | H1056 |
| 010614.06 | BXD39 | CD | F | 730 | 673 | H0673 |
| 102414.02 | BXD39 | CD | F | 550 | 1322 | H1322 |
| 121214.22 | BXD39 | HF | F | 350 | 1474 | H1474 |
| 121214.21 | BXD39 | HF | F | 350 | 1473 | H1473 |
| 061913.06 | BXD40 | CD | F | 578 | 680 | H0680 |
| 121214.13 | BXD40 | HF | F | 358 | 1593 | H1593 |
| 111314.05 | BXD40 | HF | F | 291 | 1488 | H1488 |
| 102414.08 | BXD40 | CD | F | 180 | 1691 | H1691 |
| 102414.12 | BXD44 | HF | F | 715 | 1687 | H1687 |
| 012615.15 | BXD44 | CD | F | 379 | 1505 | H1505 |
| 012615.12 | BXD44 | HF | F | 379 | 1501 | H1501 |
| 042015.07 | BXD44 | CD | F | 240 | 1822 | H1822 |
| 042015.10 | BXD44 | HF | F | 240 | 1825 | H1825 |
| 121515.12 | BXD45 | CD | F | 507 | 1826 | H1826 |
| 121515.15 | BXD45 | HF | F | 503 | 1940 | H1940 |
| 012615.11 | BXD45 | CD | F | 384 | 1762 | H1762 |
| 042915.13 | BXD48 | HF | F | 595 | 1377 | H1377 |
| 121515.18 | BXD48 | CD | F | 518 | 1835 | H1835 |
| 042015.24 | BXD48 | HF | F | 189 | 2046 | H2046 |
| 042015.27 | BXD48 | CD | F | 188 | 2054 | H2054 |
| 062013.06 | BXD48a | HF | F | 543 | 699 | H0699 |
| 051112.05 | BXD48a | CD | F | 233 | 209 | H0209 |
| 051112.04 | BXD48a | HF | F | 233 | 210 | H0210 |
| 010614.07 | BXD53 | HF | F | 713 | 833 | H0833 |
| 062013.16 | BXD53 | CD | F | 571 | 848 | H0848 |
| 021213.14 | BXD60 | CD | F | 530 | 171 | H0171 |
| 050912.08 | BXD60 | CD | F | 235 | 235 | H0235 |
| 121515.23 | BXD61 | CD | F | 524 | 1841 | H1841 |
| 121515.25 | BXD61 | HF | F | 524 | 1843 | H1843 |
| 111414.02 | BXD61 | CD | F | 188 | 1770 | H1770 |
| 111414.03 | BXD61 | HF | F | 188 | 1922 | H1922 |
| 111414.04 | BXD61 | HF | F | 188 | 1722 | H1722 |
| 082214.10 | BXD62 | HF | F | 548 | 1315 | H1315 |
| 042915.19 | BXD62 | HF | F | 488 | 1434 | H1434 |
| 121214.20 | BXD62 | CD | F | 353 | 1436 | H1436 |
| 042015.05 | BXD62 | CD | F | 253 | 1847 | H1847 |
| 102616.17 | BXD63 | HF | F | 752 | 2091 | H2091 |
| 083016.03 | BXD63 | CD | F | 751 | 1872 | H1872 |
| 121214.24 | BXD63 | CD | F | 344 | 1414 | H1414 |
| 121214.26 | BXD63 | HF | F | 344 | 1416 | H1416 |
| 090412.08 | BXD63 | CD | F | 218 | 818 | H0818 |
| 111414.09 | BXD63 | HF | F | 186 | 1715 | H1715 |
| 121615.09 | BXD65 | CD | F | 541 | 1787 | H1787 |
| 121615.06 | BXD65 | HF | F | 541 | 1784 | H1784 |
| 051012.01 | BXD65 | HF | F | 230 | 458 | H0458 |
| 042015.13 | BXD65 | CD | F | 222 | 2098 | H2098 |
| 121615.12 | BXD65b | CD | F | 527 | 1793 | H1793 |
| 121615.11 | BXD65b | HF | F | 527 | 1790 | H1790 |
| 111414.05 | BXD65b | CD | F | 187 | 1766 | H1766 |
| 102414.18 | BXD65b | HF | F | 174 | 1713 | H1713 |
| 121214.04 | BXD66 | HF | F | 367 | 1560 | H1560 |
| 121214.19 | BXD66 | CD | F | 354 | 1558 | H1558 |
| 042415.08 | BXD66 | CD | F | 184 | 2128 | H2128 |
| 042415.05 | BXD66 | HF | F | 184 | 2124 | H2124 |
| 082214.02 | BXD68 | CD | F | 545 | 1080 | H1080 |
| 082214.08 | BXD68 | HF | F | 545 | 1290 | H1290 |
| 101613.05 | BXD68 | CD | F | 201 | 1068 | H1068 |
| 101613.08 | BXD68 | HF | F | 201 | 1071 | H1071 |
| 061913.13 | BXD69 | CD | F | 558 | 591 | H0591 |
| 121214.23 | BXD69 | HF | F | 528 | 1328 | H1328 |
| 101613.09 | BXD69 | HF | F | 210 | 1087 | H1087 |
| 042415.23 | BXD69 | CD | F | 157 | 2142 | H2142 |
| 121615.14 | BXD70 | HF | F | 578 | 1728 | H1728 |
| 121214.16 | BXD70 | CD | F | 357 | 1512 | H1512 |
| 050115.06 | BXD70 | CD | F | 261 | 1855 | H1855 |
| 051012.08 | BXD70 | HF | F | 239 | 188 | H0188 |
| 121615.17 | BXD73 | CD | F | 539 | 1906 | H1906 |
| 121214.10 | BXD73 | CD | F | 361 | 1451 | H1451 |
| 121214.11 | BXD73 | HF | F | 361 | 1447 | H1447 |
| 042015.18 | BXD73 | HF | F | 206 | 2071 | H2071 |
| 050115.15 | BXD73b | CD | F | 497 | 1466 | H1466 |
| 050115.12 | BXD73b | HF | F | 474 | 1545 | H1545 |
| 051112.12 | BXD73b | CD | F | 238 | 184 | H0184 |
| 051112.14 | BXD73b | HF | F | 237 | 183 | H0183 |
| 121615.19 | BXD77 | HF | F | 511 | 1866 | H1866 |
| 050115.17 | BXD77 | CD | F | 460 | 1398 | H1398 |
| 061913.18 | BXD79 | HF | F | 571 | 583 | H0583 |
| 050115.19 | BXD79 | CD | F | 468 | 1555 | H1555 |
| 090412.10 | BXD79 | CD | F | 217 | 825 | H0825 |
| 061913.19 | BXD87 | CD | F | 581 | 296 | H0296 |
| 121615.21 | BXD87 | HF | F | 512 | 1945 | H1945 |
| 051012.14 | BXD87 | HF | F | 241 | 243 | H0243 |
| 090612.10 | BXD87 | CD | F | 200 | 550 | H0550 |
| 101014.04 | BXD89 | HF | F | 539 | 1321 | H1321 |
| 051112.01 | BXD89 | HF | F | 243 | 176 | H0176 |
| 090612.14 | BXD89 | CD | F | 193 | 555 | H0555 |
| 062013.03 | BXD90 | HF | F | 570 | 736 | H0736 |
| 062013.02 | BXD90 | CD | F | 549 | 756 | H0756 |
| 101613.19 | BXD90 | HF | F | 218 | 1093 | H1093 |
| 102414.03 | BXD90 | CD | F | 188 | 1702 | H1702 |
| 062013.17 | BXD91 | CD | F | 562 | 883 | H0883 |
| 062013.19 | BXD91 | HF | F | 562 | 881 | H0881 |
| 102414.20 | BXD95 | HF | F | 550 | 1273 | H1273 |
| 101014.10 | BXD95 | CD | F | 536 | 1742 | H1742 |
| 101613.21 | BXD95 | HF | F | 213 | 1275 | H1275 |
| 012615.17 | BXD98 | CD | F | 384 | 1769 | H1769 |
| 012615.16 | BXD98 | CD | F | 384 | 1524 | H1524 |
| 111414.14 | BXD99 | CD | F | 535 | 1354 | H1354 |
| 050914.05 | BXD99 | HF | F | 182 | 1355 | H1355 |
| 111314.15 | BXD100 | CD | F | 567 | 1190 | H1190 |
| 050914.04 | BXD100 | HF | F | 537 | 1187 | H1187 |
| 051112.07 | BXD100 | CD | F | 223 | 247 | H0247 |
| 051112.08 | BXD100 | CD | F | 223 | 248 | H0248 |
| 042415.12 | BXD100 | HF | F | 176 | 2085 | H2085 |
| 102616.20 | BXD101 | CD | F | 756 | 2114 | H2114 |
| 042715.19 | BXD101 | HF | F | 493 | 1422 | H1422 |
| 111414.20 | BXD101 | CD | F | 349 | 1425 | H1425 |
| 051112.09 | BXD101 | CD | F | 223 | 468 | H0468 |
| 042915.01 | BXD102 | CD | F | 501 | 1499 | H1499 |
| 121214.07 | BXD102 | HF | F | 366 | 1498 | H1498 |
| 053014.01 | BXD102 | CD | F | 183 | 1363 | H1363 |
| 052814.04 | BXD102 | HF | F | 183 | 1361 | H1361 |
About tissue
Tissue collection started at approximately 9 am with the anesthetic Avertin used via intraperitoneal injection of 0.2 mL per 10 g of body weight. Animals were perfused with ice-cold phosphate-buffered saline. The liver was the first organ harvested. The gall bladder was removed, the liver weighed, and then immediately frozen in liquid nitrogen in 20 mL scintillator vials. Roughly 20 mg of pre-pulverized liver tissue was mixed with 1 mL of TRIzol reagent at 4°C for RNA extraction. The sample was then further homogenized in TRIzol with a metallic bead for 2x30s at 25 Hz. The homogenate was transferred to a new tube (without the bead) and 200 µL of chloroform was added and mixed. Samples were centrifuged at 12,000g for 15 minutes at 4°C. ~400 µL of the clear top phase was taken and added to a tube with 400 µL isopropanol, followed by vortexing and the same centrifugation. The RNA pellet was observed and the liquid was discarded. The RNA pellet was resuspended in ethanol by pipetting, then the samples were centrifuged again. The ethanol was removed and the sample was air dried and quantified by Nanodrop. Next, samples were cleaned up using the RNEasy MinElute kits (Qiagen) as per manufacturing instructions.
About platform
RNA-seq and RNA integrity (RIN) checks were performed by an Agilent 2100 Bioanalyzer, and samples with RIN ≥ 6 were retained for RNA-seq, which was run on a NovoGene HiSeq 3000 with 150 bp end paired reads after polyA+ enrichment at the University of Tennessee Health Science Center transcriptomics platform. Samples were measured with an average of 24±2.7 million reads (mean±standard deviation).
About data processing
Raw fastq files were aligned to the reference mouse genome using STAR version 2.6.0c, using the UCSC genome assembly version GRCm38 (mm10). An average of 86.4±2.6% of reads were mapped per sample. Reads were counted using RSeQC version 2.6.4. Read counts were normalized to RPKM values using gene lengths from ENSEMBL82 v2015-10-02. All RNA-seq data were then scaled by adding 1 to the normalized counts and then taking the log2.