 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 30, 2019 |
| Title |
control-A rep5 [437A] |
| Sample type |
SRA |
| |
|
| Source name |
control-A
|
| Organism |
Bos taurus |
| Characteristics |
breed: Holstein heifers
age: 11-13 months
Sex: female
location: University of Florida Dairy Unit
treatment: control vehicle
tissue: ampulla
|
| Treatment protocol |
The animals were clinically healthy prior to the experiment, tested negative for Brucella abortus, Neospora caninum and Leptospirosa, and had clear vaginal mucus. Estrous cycles were synchronized using a modified 5-d Co-synch protocol and were injected intramuscularly with 100 mg gonadotropin-releasing hormone (GnRH; gonadorelin diacetate tetrahydrate) followed by 25 mg prostaglandin F2α (dinoprost tromethamine) 5 and 6 days later, and 100 mg GnRH 8 days later. Starting the day after the final GnRH injection, animals were injected intramuscularly with 200 mg progesterone in corn oil (50 mg/mL) daily for 7 d to ensure elevated circulating progesterone concentrations at the time of intrauterine infusion. Three days after the final GnRH injection, animals were blocked by age and weight, and randomly assigned to intrauterine infusion of control sterile medium (n = 6), using 30 ml Luria-Bertani broth, or bacteria (n = 4), using 4.64 × 10^7 CFU/mL E. coli MS499 and 3.38 × 10^7 CFU/mL T. pyogenes MS249 in 30 mL Luria-Bertani broth. Vaginal mucus was collected daily using a Metricheck device from the day before until 7 days after infusion, and endometritis was graded as described previously; grade 0, clear or translucent mucus; grade 1, mucus containing flecks of pus; grade 2, mucus containing ≤ 50% pus; and grade 3, mucus containing > 50% pus. Clinical endometritis was induced 4 to 6 days after infusion in the bacterial-infusion animals but not in controls as evidenced by pus detectable in the vagina (median endometritis grade 3 vs. ≤ 1).
|
| Growth protocol |
To avoid confounding effects of periparturient problems, lactation and metabolic stress, we used virgin Holstein heifers, 11 to 13 months old.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Two weeks prior to slaughter, the oestrous cycles of all the animals were synchronized using the modified 5-d Co-synch protocol described above. Animals were slaughtered by captive bolt and exsanguination 94 days after infusion. Reproductive tracts were collected, transported on ice, and processed within 1 h of slaughter. Endometrial tissue was dissected away from underlying myometrium, and oviduct samples were collected by extrusion into a collection vessel. Granulosa cells were isolated from dominant follicles > 8 mm diameter by aspiration, using a sterile 20-gauge needle and syringe containing collection medium (0.5% BSA, 20 mM HEPES, 2 mM sodium pyruvate, 10 IU/ml heparin, 100 U/ml penicillin, 100 ug/ml streptomycin in Medium 199); the aspirates were centrifuged at 500 × g to isolate the granulosa cells. Samples of caruncular and intercaruncular endometrium, oviduct isthmus and ampulla, and granulosa cells were snap frozen in liquid nitrogen and stored at -80°C. Technical problems prevented collection of the oviduct from one bacteria-infused animal and the intercaruncular endometrium from another bacteria-infused animal
Samples were thawed, suspended in RLT buffer, and RNA extracted using the RNeasy Micro kit. To produce RNAseq libraries, 500 ng (endometrium and oviduct) or 200 ng (granulosa) total RNA was used for mRNA isolation using NEBNext Poly(A) mRNA Magnetic Isolation Module, followed by RNA library construction with NEBNext Ultra II Directional Library Preparation (both New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions. Briefly, extracted mRNA was fragmented in NEBNext first-strand synthesis buffer at 94°C, followed by first-strand cDNA synthesis using reverse transcriptase and random primers. Synthesis of double-stranded cDNA was performed using the second strand master mix provided with the kit. The resulting double-stranded cDNA was end-repaired, dA-tailed and ligated with NEBNext adaptors. Finally, libraries were enriched by amplification and purified by Meg-Bind RxnPure Plus beads (Omega Biotek, Norcross, GA, USA). Barcoded libraries were sized on a bioanalyzer, and quantified by Qubit and qPCR. All individual libraries were pooled, and equal molar concentrations were run on an Illumina HiSeq3000 sequencer. Both library construction and sequencing were performed at the ICBR of the University of Florida
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
Illumina basespace used for basecalling.
Reads acquired from the sequencing platform were cleaned with the Cutadapt program (Martin 2011) 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.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
The transcripts of Bos taurus (76,341 sequences) retrieved from the NCBI genome database (GCF_002263795.1) were used as reference sequences 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 (Langmead and Salzberg 2012). The mapping results were processed with the SAMtools (Li et al. 2009), and scripts developed in-house by the Interdisciplinary Centre for Biotechnology Research to remove potential PCR duplicates and select uniquely-mapped reads for gene expression analysis, as described previously (Piersanti et al. 2019a). Gene expression was compared between the bacteria-infused and control animals by counting the number of mapped reads for each transcript (Yao and Yu 2011).
Ingenuity Pathway Analysis (Qiagen) was used to identify pathways, gene networks, and upstream regulators of DEGs affected by bacterial infusion (Krämer et al. 2014). Pathways were identified by -log P > 1.3 and log2FC ≤ −2 or ≥ 2, and corresponding z-scores were calculated to predict activation status. Gene networks were identified by assessing the number of DEGs in each network, and gene network scores were calculated by the software (a network score of ≥ 2 gives 99% confidence the network was not identified by chance). Upstream regulators of DEGs, and predicted diseases and functions were identified by z-scores ≥ 2 or ≤ −2, and were considered significant predictors of activation or inhibition of DEGs, respectively
Genome_build: GCF_002263795.1
Supplementary_files_format_and_content: tab-delimited text files include the mapped read counts for each Sample
|
| |
|
| Submission date |
Nov 15, 2019 |
| Last update date |
Dec 01, 2019 |
| Contact name |
FAHONG YU |
| E-mail(s) |
[email protected]
|
| Phone |
3522738064
|
| Organization name |
UNIVERSITY OF FLORIDA
|
| Department |
ICBR
|
| Street address |
2033 Mowry Rd
|
| City |
GAINESVILLE |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
| |
|
| Platform ID |
GPL21659 |
| Series (1) |
| GSE140469 |
Uterine infection alters bovine endometrium, oviduct and granulosa transcriptome three months later |
|
| Relations |
| BioSample |
SAMN13292387 |
| SRA |
SRX7156824 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4162530_437A_count.txt.gz |
125.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
