19_Bis_SC30
Homo sapiens
Protocols: In total, 48 blastocyst embryos (Day-5/6/7) from 27 unrelated couples were collected after a PGT analysis concluding in an affected status, performed in PCBN. Among them, 38 were affected by a non-metabolic, non-mitochondrial genetic disorder and considered as a “control” group (Table 1), and 10 were affected by a mitochondrial disorder (“mitochondrial” group). In the mitochondrial group, 9 embryos carried a mtDNA mutation (m.3243A>G n=2, m.8344A>G n=3 and m.8993T>C n=4), and one embryo was homozygous for the c.352C>T; p.(Arg118Cys) variant in the DGUOK nuclear gene (NM_080916.3). Heteroplasmy levels (Table 1) were assessed independently on one or two blastomeres sampled at day 3, by using the semi-quantitative fluorescent PCR method followed by enzymatic digestion, as already described (Gigarel et al., 2005 and Monnot et al., 2011). All embryos were vitrified when they have reached the blastocyst stage by day-5, day-6 and/or day-7 of development (RapidVitTM Blast, Vitrolife, Paris, France) before RNA-Sequencing analyses. Embryos were scored according to their quality into three categories (good, fair or poor), based on the guidelines of embryonic morphological assessment (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). The day of embryo collection, blastocyst stage embryos (Day-5/6/7) were warmed (RapidWarmTMBlast, Vitrolife, Paris, France) and re-expanded under culture conditions (37oC, 5% O2, 5% CO2). For embryos that haven't hatched, zona pellucida drilling was carried out through laser pulses under the microscope micromanipulator. Embryos were subsequently rinsed in a drop of PBS 1X and collected in separate DNA LoBind tubes (Eppendorf, Montesson, France), containing cell lysis buffer and murine RNase inhibitor, using the NEBNext Single Cell/ Low Input RNA Library Prep Kit for Illumina (New England Biolabs, Evry, France). Lysed embryos were stored at -80oC for a short-term period, to proceed with the RNA-Sequencing protocol in the next 10 days. RNA-Sequencing libraries' starting material was based on the number of embryonic cells at the blastocyst stage, which corresponded to approximately 100 cells (~100 pg of RNA). cDNA synthesis from total RNA was performed using the NEBNext Single Cell/ Low Input RNA Library Prep Kit for Illumina (New England Biolabs, Evry, France), according to the manufacturer's guidelines. Primer annealing and first strand synthesis was carried out using a reverse transcription (RT) primer mix and reverse transcriptase. “SMART” strategy by template switching was included for converting mRNA molecules into full length cDNA. Second strand synthesis and cDNA amplification were performed by DNA polymerase. The cDNA was purified using the AMPureXP Beads (Beckman Coulter, Villepinte, France) and was then sheared by enzymatic digestion. Libraries were prepared according to the manufacturer's instructions. Library quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Paris, France) and concentration measured with a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Villebon sur Yvette, France). Samples with amplification bias, identified by an unexpected library distribution on Bioanalyzer, were excluded from subsequent analysis. When samples presented a primer dimer peak in the Bioanalyzer trace, the cleanup step of the PCR reaction was repeated.
ENA-FIRST-PUBLIC
2023-02-27
ENA-LAST-UPDATE
2023-02-27
External Id
SAMEA13327899
INSDC center alias
Plateforme Bio-informatique, Universite Paris Descartes, Structure Federative de Recherche Necker, INSERM US24/CNRS UAR 3633, Paris, France
INSDC center name
Plateforme Bio-informatique, Universite Paris Descartes, Structure Federative de Recherche Necker, INSERM US24/CNRS UAR 3633, Paris, France
INSDC first public
2023-02-27T00:14:39Z
INSDC last update
2023-02-27T00:14:39Z
INSDC status
public
Submitter Id
E-MTAB-11531:19_Bis_SC30
age
5 to 7
broker name
ArrayExpress
common name
human
developmental stage
embryo
disease
normal
genotype
wild type genotype
organism part
blastocyst
sample name
E-MTAB-11531:19_Bis_SC30
scientific_name
Homo sapiens