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Sample GSM7060869 Query DataSets for GSM7060869
Status Public on May 03, 2023
Title Vitrified in Metaphase II - VMAT-3
Sample type SRA
Source name Equine oocytes
Organism Equus caballus
Characteristics cell type: Equine oocytes
treatment: VMAT
Treatment protocol Vitrification was performed on immatured oocytes surrounded by corona radiata, warmed and in vitro matured, then denuded, polar body evaluated and frozen for RNA extraction (VGV group), this was compared with oocytes in vitro matured, denuded, polar body visualized, vitrified and warmed and frozen for transcriptomics (VMAT group) and compared with fresh oocytes, in vitro matured, denuded and polar body evaluated before being frozen for RNA extraction.
Growth protocol Routine in vitro maturation was used for equine embryo production, as described previously by (Papas et al, Animals (Basel). 2021 Jul; 11(7)). During in vitro maturation, an average of 20 cumulus oocyte complexes were cultured in 500-µL of maturation medium (Medium 199 with Earl’s salts (Gibco) containing 10% (v/v) FBS (Gibco), 9.4 μg/mL folli-cle-stimulating hormone, and 1.88 μg/mL luteinizing hormone (Stimufol, Reprobiol, Ouffet, Belgium) and 50-µL gentamacyn ) covered with Paraffin oil (SAGE oil for tissue culture, ART-4008-5P, Cooper Surgical Company), at 38.5 °C in 5% CO2 in air .
Extracted molecule total RNA
Extraction protocol Three groups of equine oocytes in matured stage were collected (equine oocytes after vitrification – warming – and in vitro maturation (VGV), in vitro maturation – vitrification – warming (VMAT), and fresh in vitro matured oocytes (FR). For each group, total RNA was isolated from 5 replicates of average 23 oocytes using the RNeasy Micro kit (Qiagen) according to the manufacturer's protocol. The quality and concentration of the RNA samples were examined using an RNA 6000 Pico Chip (Agilent Technologies) and a Quant-iT RiboGreen RNA Assay kit (Life Technologies), respectively.
Transcriptome library preparation was done by Qiaseq UPX 3' transcriptome kit (Qiagen)
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description C19
Data processing Quality trimming and adapter removal, trim_galore --stringency 3 --length 25 -a AGATCGGAAGAGC -a AAAAAAAAAA " --output_dir <out_dir> <in.fastq.gz>
Mapping reads with STAR, STAR --genomeDir <genome_dir> --readFilesIn <trimmed.fastq.gz> --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.6 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMattributes NH HI NM MD --outSAMtype BAM Unsorted --outReadsUnmapped Fastx --outFileNamePrefix star/<sample_name> --quantMode TranscriptomeSAM
Samtools sort, samtools sort -o <out_sorted.bam> <star.bam>
Deduplication of the UMIs, umi_tools dedup -I <bam>
Samtools sort on name, samtools sort-n -o <out.bam> <in.bam>
Quantify with rsem, rsem-calculate-expression --forward-prob 1 --no-bam-output --bam <in.bam> <index_rsem>
Assembly: Equus_caballus.EquCab3.0 release 108 Ensembl
Supplementary files format and content: normalized_counts_deseq2.csv: comma-delimited text file (csv) contains normalized gene expression counts with DESeq2's standard method
Submission date Feb 23, 2023
Last update date May 05, 2023
Contact name Katrien Smits
Organization name Ghent University
Department Internal Medicine, Reproduction and Population Medicine
Street address Salisburylaan 133
City Merelbeke
ZIP/Postal code 9820
Country Belgium
Platform ID GPL21401
Series (1)
GSE225950 Transcriptomics reveals molecular differences in equine oocytes vitrified before and after in vitro maturation
BioSample SAMN33426156
SRA SRX19487383

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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