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| Status |
Public on Mar 22, 2019 |
| Title |
treatment rep5 |
| Sample type |
SRA |
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| Source name |
blood
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| Organism |
Bos taurus |
| Characteristics |
tissue: blood
location: University of Florida Dairy Unit
age: lactation, female
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| Treatment protocol |
The dominant follicle was sampled 63 ± 3 d post partum following a caudal epidural injection of 60 mg of lidocaine hydrochloride 2% (Aspen Veterinary Resources, Greely, CO, USA). The perineum and vulva were cleaned and disinfected with povidone iodine followed by 70% ethanol. Vagina lavage with 100 mL of 0.2% chlorohexidine was followed by two washes with 100 mL sterile 0.9% saline. An oocyte pick-up instrument including a 7.5 MHz convex ultrasound probe (Choice Medical, South Pasadena, FL, USA) was covered in a sanitary sleeve (IMV Technologies, Normandy, France) and introduced into the vagina with sterile lubricant. The ovary was visualized by ultrasound (Aloka SSD-500, Hitachi Healthcare Americas, Twinsburg, OH, USA), and the internal diameter of the dominant follicle was measured based on the average of two measurements at 90° angle of the diameters from the larger cross sectional area of the follicle. An 18 gauge needle attached to a tygon tube connected to a vacuum pump was introduced into the oocyte pick-up instrument and the dominant follicle aspirated into collection medium (Medium 199 with Earle’s Salts, 0.5% BSA, 20 mM HEPES, 2 mM sodium pyruvate, 10 IU/mL heparin 100 U/mL penicillin and 100 μg/mL streptomycin; all Fisher Scientific, Hampton NH). The needle was maintained inside the follicle and 10 mL of collection medium was infused through the needle to flush the follicle. The volume of aspirate was recorded prior to centrifugation at 300 ₓ g for 10 min to separate the follicular fluid from granulosa cells, which were washed twice in sterile PBS. The follicular fluid and granulosa cells were stored at -80°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Granulosa cells were thawed and resuspended in RLT buffer for RNA extraction using the RNeasy Micro kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Total RNA concentration was determined using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA with a 28S to 18S ratio > 1 and RNA integrity number (RIN) ≥ 7 were used for RNAseq library construction.
To produce RNAseq libraries, 200 ng of total RNA was used to isolate mRNA using NEBNext Poly(A) mRNA magnetic isolation module (New England Biolabs, Ipswich, MA, USA) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions. Briefly, extracted mRNA was fragmented in NEBNext first strand synthesis buffer by heating at 94°C, followed by first strand cDNA synthesis using reverse transcriptase and random primers. Synthesis of double-stranded cDNA was performed using the 2nd strand master mix provided with the kit. The resulting double-stranded cDNA was end-repaired, dA-tailing and ligated with NEBNext adaptors. Finally, libraries were enriched by 12 cycles of amplification and purified by Meg-Bind RxnPure Plus beads (Omega Biotek, Norcross, GA, USA).
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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 (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.
The transcripts of Bos taurus (80,896 sequences) retrieved from the NCBI RefSeq database 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 & Salzberg 2012). The mapping results were processed with the samtools and scripts developed in house at the University of Florida ICBR to remove potential PCR duplicates and choose uniquely mapped reads for gene expression analysis. Gene expression between the control and treatment group was assessed by counting the number of mapped reads for each transcript (Yao & Yu 2011).
Pathway analysis was performed using Ingenuity Pathway Analysis (Qiagen). Differentially expressed genes with a P-value ≤ 0.05 and log2 FC < -2 or > 2 were used for analysis. 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 differentially expressed genes in a given gene network. Upstream regulators of specific gene networks and upstream regulators of differentially expressed genes were predicted using IPA algorithms.
Genome_build: btaurus_ref
Supplementary_files_format_and_content: tab-delimited text files include the mapped read counts for each Sample
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| Submission date |
Mar 21, 2019 |
| Last update date |
Mar 22, 2019 |
| 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) |
| GSE128697 |
Persistent effects on bovine granulosa cell transcriptome after resolution of uterine disease |
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| Relations |
| BioSample |
SAMN11229762 |
| SRA |
SRX5557499 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3682876_9234.txt.gz |
55.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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