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| Status |
Public on Aug 03, 2022 |
| Title |
581_D |
| Sample type |
SRA |
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| Source name |
Precision-cut bovine liver slices
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| Organism |
Bos taurus |
| Characteristics |
tissue: Liver
treatment: PPARD agonist
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| Treatment protocol |
). Within the plate, PCLS were treated with 100 µM of the PPARα agonist WY-14643 (CATNO,PRODUCT,PLACE), 50 µM of the PPARδ agonist GW-501516 (ALX-420-032-M005, Enzo, NY, USA), or 100 µM of the PPARγ agonist rosiglitazone (R0106, TCI, OR, USA); 200 µM of palmitic acid (100905, MP Biomedicals, CA, USA) or 100 µM of serum NEFA, isolated via solid phase extraction as previously reported [145]. WY-14643 concentration was selected based on prior reports [156]; additionally, according to our findings in an immortalized model of bovine liver [145], the dose-dependent response to PPARα and PPARγ is similar (hence the 100 µM concentration of both WY and rosiglitazone), while PPARδ modulation was about twice as sensitive (hence the 50 µM dose for GW-501516). Additionally, peak PPAR activation for palmitic acid was achieved at 200 µM. The concentration of NEFA was chose to mimic the physiological concentration of NEFA in the lactating dairy cow. In Plate 3, PCLS from each cow were cultured in blood serum isolated from the same cow on the day of the liver biopsy; PPAR antagonists were added at 50 µM in duplicates to the wells: for PPARα, GW-6471 (9453, CAS# 880635-03-0, BioVision incorporated, CA, USA); for PPARδ, GSK-3787 (3961/10, CAS# 188591-46-0, Tocris, Bio-Techne Corporation, MN, USA); for PPARγ, GW-9662 (70785, CAS# 22978-25-2, Cayman Chemicals, MI, USA). Additionally, palmitic acid and serum NEFA were also supplemented with doses as in Plates 1 and 2. All treatments were adjusted for the vehicle (DMSO, D2438, Millipore Sigma, MO, USA) at a volume of 0.8%. Plates were placed in a modular incubator chamber (MIC-101, Billups-Rothenberg, CA, USA), clamped shut, and flushed with carbogen (95 % 02, 5% CO2) for 10 minutes. The modular chamber was placed inside a cell culture incubator, and atop a benchtop orbital shaker located inside of the incubator. The slices were incubated for 18 hours at 37 C, ~100 rpm.
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| Growth protocol |
PCLS were prepared following the protocol developed by De Graaf and collaborators [155], with few modifications. Briefly, liver tissue samples were transported to the laboratory in ice-cold KHB; upon arrival, they were immediately embedded in low gelling ultrapure agarose (16500-100, Invitrogen, CA, USA) inside a 8 mm mold-plunger assembly (MD2200, tissue embedding unit, Alabama Research & Development, AL, USA). The plunger with the embedded tissue was then transferred to a Krumdieck Tissue Slicer (MD1000-A1, Alabama Research & Development, AL, USA), pre-filled with ice-cold KHB; slice thickness was set at 280-300 µm, with a cycle speed of ~35 and using the “intermittent blade mode”. Slices were then collected and placed on a new tissue culture dish, prefilled with a minimum amount of cold KHB to prevent dehydration. A total of 36 PCLS were selected for each animal at each timepoint, evaluating visually to identify those with the best-defined shape, and without holes or patent morphological irregularities. The 36 PCLS were thus transferred to three 12-well culture plates (665180, Greiner Bio One, NC, USA) and the treatment were supplied in duplicates. In Plates 1 and 2, PCLS were cultured in William’s Medium E (WME, A1217601, Gibco, Thermo Fisher Scientific, MA, USA), supplemented with GlutaMAX (35050-061, Gibco, NY, USA), 14 mM of D-glucose monohydrate (0643-1KG, VWR, OH, USA), and 50 µg/mL of gentamycin (15750060, Life Technologies, OR, USA).
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| Extracted molecule |
total RNA |
| Extraction protocol |
PCLS from Plates 2 and 3 were collected after incubation, and transferred to separate screw-cap vials (490003-520, VWR, PA, USA), pre-filled with 600 µL of ice-cold TRIzol reagent (15596026, Thermo Scientific, MA, USA) and two 3.2 mm beads, homogenized using a Geno/Grinder Automated Tissue Homogenizer (2010-115, SPEX SamplePrep, NJ, USA; courtesy of the Department of Crop and Soil Sciences, Oregon State University, OR, USA), disrupting the tissue in short burst (45 seconds) at 1500 rpm, followed by incubation on ice for 3 minutes; the disruption-incubation cycle was repeated up to three times, or until no tissue pieces were visible within the tubes. Immediately after disruption, 120 µL of pre-chilled chloroform was added the tube, and the samples were mixed by inverting the tube, and incubated on ice for 5 minutes. After incubation, the samples were transferred to a new 1.7 mL microcentrifuge tube, and centrifuged a 4 C for 15 minutes, 15,000 x g. The upper-phase supernatant (~200 µL) was collected and RNA was purified using a Mag-MAX-96 Total RNA Isolation Kit (AM1830, Invitrogen, MA, USA) following the manufacturer’s instructions, with minor modifications: briefly, 100 µL of each sample was transferred to the first row (A) of a 96-Well DeepWell Storage Plate (260251, Thermo Scientific, MA, USA). Rows B-F contained reagents supplied with the kit: 20 µL of the magnetic beads mix (row B), 150 µL of wash solution 1 (row C), 150 µL of wash solution 2 (row D), and 50 µL of DNAse-RNAse free water (VWRL0201-0500, VWR, PA, USA) in rows E and F. A suitable protocol was then generated to mimic the manufacturer’s protocol with a KingFisher Duo Purification System (5400110, Thermo Scientific, MA, USA). Eluted RNA was measured using a SpectraDrop Micro-Volume Microplate in a SpectraMax plus 384 spectrophotomoter (89212-396, Molecular Devices, CA, USA). Average 260/230 and 260/280 ratios were 2.06 ± 0.24 and 1.69 ± 0.27, respectively. RNA integrity was assessed by the Center for Genome Research and Bioinformatics at Oregon State University using an Agilent Bioanalyzer 2100 (G2939BA, Agilent, CA, USA). For one animal, +10 DIM PCLS were excluded from the analysis, as their RIN was below 3; unsurprisingly, these samples clustered together in the principal component analysis of RNAseq results and were very dissimilar from the other animals, which further justified their removal; with that sample removed, resulting RNA integrity numbers were 7.26 ± 0.64.
PCLS from postpartum animals treated with the three isotype-specific PPAR agonists, as well as the control group, were sent to the Center for Genome Research and Bioinformatics at Oregon State University for high-throughput sequencing. Library construction was obtained using a QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina (015.96, Lexogen, NH, USA). Sequencing was performed on an Illumina HiSeq3000 platform, at 60 samples/lane. The raw reads have been deposited
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
lane1-s018-index-ACGTCT-21_581_D_-rd_S18
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| Data processing |
Quality control was assayed using MultiQC v1.8 [158] (https://multiqc.info/). Reads were then trimmed based on PHRED score and adapter presence using TRIMMOMATIC v0.39 [159](https://github.com/usadellab/Trimmomatic) with arguments LEADING:5 TRAILING:5 SLIDINGWINDOW:4:5 MINLEN:3
A genome index was generated using the ARS-UCD1.2 Bos taurus genome (http://ftp.ensembl.org/pub/release-104/fasta/bos_taurus/dna/) and the ARS-UCD1.2.104 annotation (http://ftp.ensembl.org/pub/release-104/gtf/bos_taurus/), using the genomeGenerate function of STAR v2.7.1 [160](https://github.com/alexdobin/STAR).
). Trimmed reads were aligned against the reference genomic index using STAR, and the average overall alignment rate was 85.46%.
Aligned .sam files were sorted and converted to .bam using samtools v1.0 [161](https://github.com/samtools/samtools), and gene count matrices were generated using stringtie v 2.0 (https://ccb.jhu.edu/software/stringtie/).
Differential expression was determined using the DESeq2 package, v1.30.1 [162](https://bioconductor.org/packages/release/bioc/html/DESeq2.html) in R v3.9, after filtering for low counts (any transcript with raw count ≤ 4). Three contrasts were generated (PPARA agonist vs CTR, PPARD agonist vs CTR, and PPARG agonist vs CTR) and DEG were considered significant with FDR ≤ 0.2.
Genome_build: ARS-UCD1.2 Bos taurus genome
Supplementary_files_format_and_content: liverslices_counts.xlsx contains raw counts for all samples
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| Submission date |
Aug 30, 2021 |
| Last update date |
Aug 03, 2022 |
| Contact name |
Massimo Bionaz |
| E-mail(s) |
[email protected]
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| Phone |
+12174938532
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| Organization name |
Oregon State University
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| Department |
Animal Sciences
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| Street address |
Weniger Hall
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| City |
Corvallis |
| State/province |
OR |
| ZIP/Postal code |
97331 |
| Country |
USA |
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| Platform ID |
GPL21659 |
| Series (1) |
| GSE183063 |
Assaying peroxisome proliferator-activated receptor activation in the peripartum using precision-cut bovine liver slices reveals novel putative PPAR targets in dairy cows. |
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| Relations |
| BioSample |
SAMN21081023 |
| SRA |
SRX11969154 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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