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Sample GSM7060866

Query DataSets for GSM7060866

Status

Public on May 03, 2023

Title

Vitrified in germinal vesicle-VGV-4

Sample type

SRA

Source name

Equine oocytes

Organism

Equus caballus

Characteristics

cell type: Equine oocytes
treatment: VGV

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

C16

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

E-mail(s)

katrien.smits@ugent.be

Organization name

Ghent University

Department

Internal Medicine, Reproduction and Population Medicine