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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 01, 2020 |
| Title |
treatment-4 rep3 |
| Sample type |
SRA |
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| Source name |
treatment-4
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| Organism |
Bos taurus |
| Characteristics |
tissue: Oocyte
location: University of Florida Dairy Unit
age: 11-13 months, female
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| Treatment protocol |
Estrous cycles were synchronized by administering 100 mg i.m. of GnRH (gonadorelin diacetate tetrahydrate; Ovacyst, Bayer) followed by 25 mg i.m. of PGF2α (dinoprost tromethamine; Prostamate, Bayer) administered 5 and 6 d later. Eight days following initial GnRH, heifers received a final dose of 100 mg of GnRH i.m. Starting on the day following the end of the synchronization protocol, heifers received 200 mg i.m. of progesterone in corn oil (50 mg/mL; Sigma-Aldrich, St Louis MO) daily for 7 days. Heifers were randomly assigned to receive intrauterine infusion of vehicle control (n = 6) or live pathogenic bacterial infusion (n = 4). Intrauterine infusion (day 0) was performed 3 days after the end of the synchronization protocol. Briefly, endometrial scarification preceded the intrauterine infusion of either vehicle medium (30 mL of sterile Luria-Bertani (LB) broth) or pathogenic bacteria (10 mL of 4.64 × 107 CFU/mL of Escherichia coli MS499, 10 mL of 3.38 × 107 CFU/mL Trueperella pyogenes MS249 followed by 10 mL of sterile LB broth). Ovum pick-up was performed using transvaginal ultrasound-guided follicle aspiration performed on days 4 and 60. Briefly, heifers received a caudal epidural injection of 60 mg of lidocaine hydrochloride 2% (Aspen Veterinary Resources, Greely CO), and the perineum and vulva were cleaned and disinfected with povidone followed by 70% ethanol. A vaginal lavage using 100 mL of 0.2% chlorohexidine in saline followed by two 100 mL lavages using sterile 0.9% saline were employed to wash the vagina.
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| Growth protocol |
To avoid confounding effects of periparturient problems, lactation and metabolic stress, we used virgin Holstein heifers, 11 to 13 months old.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted in oocytes of vehicle (n = 5) and bacteria (n = 3) infused heifers at day 4, and vehicle (n = 6) and bacteria (n = 3) infused heifers at day 60. Oocytes were thawed and suspended in 350 μL RLT buffer for the extraction of total RNA using the RNeasy Micro kit (Qiagen) according to the manufacturer’s instructions. Total RNA concentration was determined using a Qubit 2.0 Fluorometer (Fisher Scientific) and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA).
To produce RNAseq libraries for sequencing, 200 pg of total RNA was used for library construction using SMARTer Universal Low input RNA kit (Takara Bio, Mountain View, CA), combined with Illumina Nextera DNA Library Preparation Kit (Illumina, San Diego, CA), according to the manufacturer's instructions. Briefly, first strand cDNA was primed by a modified N6 primer (the SMART N6 CDS primer), then base-paired with additional nucleotides, creating an extended template. The reverse transcriptase then switches templates and continues transcribing to the end of the oligonucleotide, resulting in single-stranded cDNA containing sequences that are complementary to the SMARTer oligonucleotide. The SMARTer anchor sequence and the N6 sequence then served as universal priming sites for DNA amplification by PCR for 10 cycles. A total of 125 pg of cDNA was then fragmented by a tagementation reaction and adapter sequences were added to the template cDNA by PCR amplification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Illumina basespace used for basecalling.
Reads acquired from the sequencing platform were cleaned with the Cutadapt program to trim off sequencing adaptors, low quality bases, and potential errors introduced during sequencing or library preparation. Reads with a quality Phred-like score < 20 and read length < 40 bases were excluded from RNAseq analysis.
The NCBI Genome database transcripts of Bos taurus (80,896 sequences) was used as a reference for RNAseq analysis. The cleaned reads of each sample were mapped individually to the reference sequences using the bowtie2 mapper (v. 2.2.3) with a “3 mismatches a read” allowance. The mapping results were processed with the samtools and scripts developed in-house at the University of Florida Interdisciplinary Center for Biotechnology Research to remove potential PCR duplicates and choose uniquely mapped reads for gene expression analysis. Differentially expressed genes (DEGs) in bacteria infused heifers (compared to vehicle infused heifers) were identified at each time point (day 4 or day 60) by counting the number of mapped reads for each transcript. Differentially expressed genes were defined using the P-value and log2 fold change (FC) ≥ 2 or ≤ -2. Adjusted P-values ≤ 0.1 were used initially in addition to non-adjusted P-values ≤ 0.05.
Pathway analysis was performed using Ingenuity Pathway Analysis (IPA, Qiagen). Differentially expressed genes with the criteria of log2 FC ≥ 2 or ≤ -2, and a non-adjusted P-value ≤ 0.05 were used for IPA analysis. These criteria were applied in order to maintain uniformity throughout the investigation due to the disparity in the number of DEGs following false discovery rate analysis using the adjusted P-value. Represented canonical pathways with a -log P-value > 1.3 were determined with corresponding z-scores to describe predicted activation status. Represented gene networks were determined by assessing the number of DEGs in each gene network. Upstream regulators of specific gene networks and upstream regulators of DEGs were predicted using IPA algorithms. Predicted upstream regulators of DEGs were assigned an activation z-score to predict either upregulation or down regulation of various downstream DEGs. A z-score ≥ 2 or ≤ -2 was assumed to be a significant prediction of activation or inhibition, respectively.
Genome_build: GCF_002263795.1
Supplementary_files_format_and_content: tab-delimited text files include the mapped read counts for each Sample
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| Submission date |
Dec 02, 2019 |
| Last update date |
Jan 02, 2020 |
| Contact name |
FAHONG YU |
| E-mail(s) |
[email protected]
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| Phone |
3522738064
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| Organization name |
UNIVERSITY OF FLORIDA
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| Department |
ICBR
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| Street address |
2033 Mowry Rd
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| City |
GAINESVILLE |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
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| Platform ID |
GPL21659 |
| Series (1) |
| GSE141307 |
Intrauterine infusion of pathogenic bacteria persistently the transcriptome of oocytes. |
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| Relations |
| BioSample |
SAMN13447656 |
| SRA |
SRX7255936 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4200592_P_count.txt.gz |
109.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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