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Status |
Public on May 03, 2023 |
Title |
Vitrified in Metaphase II - VMAT-3 |
Sample type |
SRA |
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Source name |
Equine oocytes
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Organism |
Equus caballus |
Characteristics |
cell type: Equine oocytes treatment: VMAT
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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.
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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 .
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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)
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
C19
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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
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Submission date |
Feb 23, 2023 |
Last update date |
May 05, 2023 |
Contact name |
Katrien Smits |
E-mail(s) |
katrien.smits@ugent.be
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Organization name |
Ghent University
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Department |
Internal Medicine, Reproduction and Population Medicine
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Street address |
Salisburylaan 133
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City |
Merelbeke |
ZIP/Postal code |
9820 |
Country |
Belgium |
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Platform ID |
GPL21401 |
Series (1) |
GSE225950 |
Transcriptomics reveals molecular differences in equine oocytes vitrified before and after in vitro maturation |
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Relations |
BioSample |
SAMN33426156 |
SRA |
SRX19487383 |
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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