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Status |
Public on May 23, 2023 |
Title |
WIN_2 |
Sample type |
SRA |
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Source name |
Follicular fluid
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Organism |
Bos taurus |
Characteristics |
tissue: Follicular fluid cell type: Extracellular Vesicles season: Winter
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Extracted molecule |
total RNA |
Extraction protocol |
For EV isolation, follicular fluid samples were subjected to a series of centrifugations starting at 500 xg for 10 minutes to remove cells, followed by 3000 xg for 10 minutes to remove the cellular debris, and at 12,000 xg for 30 min to remove protein aggregates and large particles. All centrifugation steps were performed at 4 °C. The supernatant of FF samples was filtered through a 0.22 µM sterile filter to remove particles larger than 200 nm. For the EV isolation, 2 ml of pre-centrifuged follicular samples were subjected to an ultracentrifugation procedure at 120,000 xg for 70 min at 4 OC using the Beckman SWTi55 rotor. The EVs pellet was washed with sterile PBS and then centrifuged again at 120,000 xg for 70 min. Finally, EVs were resuspended in 500 µL of PBS and stored at −80 °C until further characterization and analysis. Total RNA including miRNAs was isolated from EVs using a Norgen Exosomal RNA Isolation kit (Norgen, Canada), according to the manufacturer’s instructions. On-column DNA digestion was performed to remove genomic DNA contaminants. Small-RNA libraries were prepared for next-generation sequencing (NGS) using a TruSeq Small RNA Library Prep Kit (Illumina) according to the manufacturer’s instructions.
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Library strategy |
miRNA-Seq |
Library source |
transcriptomic |
Library selection |
size fractionation |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
FASTQ files for each sample were generated using the bcl2fastq software (Illumina Inc.) Sequence reads were mapped to Bos Taurus reference genome (ARS-UCD1.2) using Bowtie2 (2.2.2) Mapped reads were used for annotation against bovine mature and precursor miRNAs listed in the mirBase database (release 22) Raw expression data were normalized using the trimmed mean of M-values normalization method (TMM normalization) and presented as TMM-adjusted Counts Per Million (CPM) The differential expression analysis was done using the DESeq2 statistical software package Assembly: ARS-UCD1.2 Supplementary files format and content: xlsx file include CPM values for each sample
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Submission date |
Dec 16, 2022 |
Last update date |
May 23, 2023 |
Contact name |
Ahmed Gad |
E-mail(s) |
ahmed.y.gad@agr.cu.edu.eg
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Organization name |
Colorado State University
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Department |
Biomedical Sciences
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Lab |
ARBL
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Street address |
3107 Rampart Rd
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City |
Fort Collins |
State/province |
Colorado |
ZIP/Postal code |
80521 |
Country |
USA |
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Platform ID |
GPL26012 |
Series (1) |
GSE221198 |
Ovarian follicle extracellular vesicle-microRNAs mediated response of beef cows to environmental heat stress |
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Relations |
BioSample |
SAMN32278773 |
SRA |
SRX18742235 |
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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