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Sample GSM6855680 Query DataSets for GSM6855680
Status Public on May 23, 2023
Title SUM_3
Sample type SRA
 
Source name Follicular fluid
Organism Bos taurus
Characteristics tissue: Follicular fluid
cell type: Extracellular Vesicles
season: Summer
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.
 
Library strategy miRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina NovaSeq 6000
 
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
 
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
Organization name Colorado State University
Department Biomedical Sciences
Lab ARBL
Street address 3107 Rampart Rd
City Fort Collins
State/province Colorado
ZIP/Postal code 80521
Country USA
 
Platform ID GPL26012
Series (1)
GSE221198 Ovarian follicle extracellular vesicle-microRNAs mediated response of beef cows to environmental heat stress
Relations
BioSample SAMN32278776
SRA SRX18742232

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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