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Sample GSM7018109

Query DataSets for GSM7018109

Status

Public on Feb 01, 2024

Title

pool of 10 embryos, Low glucose + BHB, replicat 3

Sample type

SRA

Source name

8-days blastocystes

Organism

Bos taurus

Characteristics

tissue: 8-days blastocystes
treatment: Low Glucose + BHB

Treatment protocol

cumulus-oocyte complexes (COCs) were divided in 50ul drops of maturation medium in groups of 10 COCs (4.5mL TCM 199 Gibco 11150-059 with 100ul/mL FBS, 40mM pyruvate, 50mg/mL gentamicin, 0,5ug FSH and 1ul/mL estradiol) between our three treatments, one control with a standard glucose concentration (5.5mM), one low glucose concentration (2.75mM) and one with low glucose concentration (2,75mM) with a supplement of BHB (1.8mM) and finally the drops are covered with mineral oil (M8410 Sigma mineral oil, SOF-BSA). The maturation medium is prepared at least 4 hours in advance and balanced in the incubator (5% CO2, 20% O2, 100% H2O) where the COCs are incubated for 24 hours.
IVF was performed using the three groups of COCs and embryos developded in the same conditions (see growth protocol)

Growth protocol

The embryos are incubated in SOF#1 until day 4. On the day 4 of development, the embryos are transferred to in drops of SOF #2 (5mL SOF Base, 0.8% BSA FAF fraction V tested (A-6003), 1X non-essential amino acids, 10uM EDTA, 1mM L-Glutamine, 0.33mM pyruvate, 0,10mM gentamicin), in which they are incubated (5% CO2, 20% O2, 100% H2O) until day 8. The embryos are divided in groups of 20-30 for each 50ul drop of SOF#2. At fay 8, embryos are washed twice in a small dish containing veterinary PBS (no calcium or magnesium) and are retrieved in 0,5mL sterile tubes with a minimal amount of PBS. They are then stored in -80C until RNA extraction,

Extracted molecule

total RNA

Extraction protocol

RNA was harvested using Simultaneous Purification of Genomic DNA and total RNA from Animal and Human Cells from the AllPrep DNA/RNA Micro Handbook (QIAGEN, USA). Each 9 samples consist of a pool of 9 to 11 embryos.
Enrichement of mRNA was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490, New England Biolabs, USA) and libraries were prepared using the NEBNext Ultra II RNA Library kit for Illumina (E7770, E7775, New England Biolabs, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

BHB3

Data processing

Raw fastq files were trimmed using fastp (with the option -w 4) then mapped to the reference genome (Bt.Ensembl105) using Kallisto.
Raw count and differential expression analysis were done using the R library DESeq2.
Assembly: bostau9
Supplementary files format and content: xlsx file containing the results of differential expression analysis between CTL vs BHB
Supplementary files format and content: xlsx file containing the results of differential expression analysis between CTL vs LG
Supplementary files format and content: xlsx file containing the results of differential expression analysis between LG vs BHB

Submission date

Feb 01, 2023

Last update date

Feb 01, 2024

Contact name

Marc-André Sirard

E-mail(s)

Marc-Andre.Sirard@fsaa.ulaval.ca

Organization name

Université Laval

Department

Sciences Animales