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| Status |
Public on Dec 31, 2021 |
| Title |
HHD_2341 [26611] |
| Sample type |
SRA |
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| Source name |
Jejunal mucosa
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| Organism |
Sus scrofa |
| Characteristics |
diet: Heart Healthy
tissue: Jejunal mucosa
rna id: 26611
breed: Ossabaw
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| Treatment protocol |
Isocaloric diets were designed to represent typical Western and heart healthy dietary patterns consumed by humans. Detailed diet composition and ingredient sources have been reported previously. Briefly, both diets contained 38% of energy (E) as fat, 47% E as carbohydrate and 15% E as protein. The major differences between the WD and HHD were the types of carbohydrate and fat, and amount of fiber, cholesterol, fruits, vegetables, and fish oil. The WD was rich in saturated fat (butter fat), cholesterol, and refined carbohydrate (sugar, white flour), and low in fiber, whereas the HHD was rich in unsaturated fat (canola, soybean and corn oils), unrefined carbohydrate (whole wheat flour, oats), fruits/vegetables (freeze dried mix, Futureceuticals, Momence, IL) and fiber, and low in cholesterol. Pigs fed the HHD were administered fish oil capsules (Epanova 1000 mg [550 mg EPA + 200 mg DHA as free fatty acids], AstraZeneca, Cambridge, MA) three times per week. Pigs in the atorvastatin (Lipitor, Pfizer, New York, NY) groups were given 20 mg/day during the first three months and 40 mg/day during the latter three months of the intervention period.
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| Growth protocol |
After one month of acclimatization to a “grower diet” and another month of a gradual shift to the experimental diets, the pigs were fed their respective diets in isocaloric amounts, with a gradual increase in total energy to meet growth requirements, for six months.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Jejunal mucosa was homogenized and RNA was isolated using TRI reagent. Isolated RNA was treated with Turbo DNase (Ambion, Waltham, MA) to minimize genomic DNA contamination. RNA quality was assessed using the Experion RNA analysis electrophoresis kit (Bio-Rad, Hercules, CA). Only samples with an RNA Quality Indicator (RQI) greater than 8 were sequenced.
Prior to RNA sequencing (RNA-seq) samples were processed (mRNA purification and fragmentation, cDNA synthesis, end repair, 3’ adenylation, adaptor ligation, and DNA enrichment) using the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA) and AMPure XP beads (Beckman Coulter, Brea, CA) per the manufacturer’s instructions. DNA fragment size was determined using Experion DNA 1K chips (Bio-Rad, Hercules, CA) and library quantification was completed using the KAPA Library Quantification kit (KAPA Biosystems, Wilmington, MA). Raw data in FASTQ format was processed for quality using CLC Bio Genomic Workbench (Qiagen, Valencia, CA). The transcriptome was assembled using the annotated Sus scrofa 11.1 as a reference genome
Samples were sequenced on an Illumina NextSeq 500 sequencer (Illumina, San Diego, CA) with 150 base pair single end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
24359 genes with unique exon read data
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| Data processing |
After sequencing, raw data in FASTQ format was processed for quality using CLC Bio Genomic Workbench (Qiagen, Valencia, CA).
The transcriptome was assembled using the annotated Sus scrofa 11.1 as a reference genome
Differential expression analysis was performed on using a two-factor model design matrix in edge R, a Bioconductor package based on a negative binomial distribution
To assess consistency of jejunal mucosa sampling, the RNA sequencing data (reads per kilobase million [RPKM]) was analyzed using principal component analysis and hierarchical clustering (Spearman ranking method; RStudio, Version 1.0.153, Boston, MA), following xCell analysis, a tool that utilizes transcriptome signals to predict enrichment of different cell types. On the basis of these analysis, one sample in the WD+S group was identified as an outlier and showed a high enrichment of preadipocytes and T-helper 1 cells relative to the other samples in the group and among all four groups, suggesting an error during sample collection. Hence, this sample was not included in subsequent analysis, resulting in a final sample size of 29 pigs. Count data on annotated genes was filtered based on a minimum of at least one count per million across six samples in all groups and normalized using the trimmed mean of M values (TMM) method. The edge R two-factor model was designed to determine differential gene expression attributable to diet, statin, or a diet x statin interaction. Differentially expressed genes were identified using the Cox-Reid profile-adjusted likelihood method and likelihood ratio test. A Benjamini-Hochberg false discovery rate (FDR) method was used to adjust p-values for multiple comparisons
Thus, given the ancillary nature of this study genes were considered differentially expressed based on an FDR adjusted p <0.2 and absolute fold change ≥1.5. Fold change for genes identified by the edge R two-factor model were interpreted as diet effect: fold change of WD relative to HHD and statin effect: fold change of atorvastatin relative to no atorvastatin. Differentially expressed genes were only attributed to main effects (diet and statin).
Genome_build: The transcriptome was assembled using the annotated Sus scrofa11.1 as a reference genome
Supplementary_files_format_and_content: comma-separated value files with Unique Exon Read counts for gene
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| Submission date |
Oct 29, 2020 |
| Last update date |
Dec 31, 2021 |
| Contact name |
Alice H Lichtenstein |
| E-mail(s) |
alice.lichtenstein@tufts.edu
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| Organization name |
Tufts University
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| Department |
Jean Mayer USDA Human Nutrition Research Center on Aging
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| Lab |
Cardiovascular Nutrition Laboratory
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| Street address |
711 Washington St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02111 |
| Country |
USA |
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| Platform ID |
GPL20983 |
| Series (1) |
| GSE160426 |
Western and Heart Healthy Dietary Patterns Differentially Affect the Expression of Genes Associated with Lipid Metabolism, Interferon Signaling and Inflammation in the Jejunum of Ossabaw Pigs |
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| Relations |
| BioSample |
SAMN16595636 |
| SRA |
SRX9400232 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4873144_PB31-lib4-2341-HH-GCCAAT_S4_R1_001_trimmed_GE_.csv.gz |
219.3 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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