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Status |
Public on Aug 31, 2019 |
Title |
PP_5 |
Sample type |
SRA |
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Source name |
Demi-blastocyst containing trophectoderm and inner cell mass cells
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Organism |
Bos taurus |
Characteristics |
pregnancy status: Pregnant at 30 and 60 days of gestation treatment during culture: Vehicle
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Treatment protocol |
Blastocysts collected on day 7 of culture were transferred individually into 50 µl drops of embryo biopsy medium (BO-biopsy, IVF Bioscience) placed on the surface of an epoxy-printed well slide to which scratches were made manually in the glass with an 18 gauge needle. Scratches were made to assist in stabilization of the embryo during the biopsy procedure. While visualizing the embryo using an inverted microscope with 20X magnification (Nikon,Diaphot, Tokyo, Japan), a micro-blade fixed to a micromanipulator was gently pressed against the middle of the embryo so that trophectoderm (TE) and inner cell mass (ICM) cells were evenly distributed on both sides (Figure 4A and B). The micro-blade was slowly moved forward and backward until the embryo was completely bisected and the zona pellucida was removed. One portion of the bisected blastocyst (the smaller or less intact portion if bisection was not uniform) was directly placed into a 0.2 ml tube containing 50 µl of RNAlater (Invitrogen, Carlsbad, CA, USA) and stored at -80°C until RNA-seq analysis. The other demi-embryo was placed into a 25 µl drop of SOF-BE2 covered in mineral oil and maintained inside an incubator at 38.5o C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2) for 30 min for re-establishment of the blastocoele after bisection. The demi-embryo was then placed into a plate containing embryo transfer medium (BO-Transfer, IVF Bioscience), loaded into a 0.25 ml embryo transfer straw, and transported to the farm in a portable incubator at 38.5°C (Micro Q Technologies).
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA from each sample was isolated using the RNeasy Micro Kit (Qiagen, Germantown, MD, USA) according to the manufacture’s cell protocol with the following adjustments for samples maintained in RNAlater; 250 µl of 100% ethanol was used instead of the recommended 350 µL of 70% (v/v) ethanol and isolated RNA was eluted in 12 µl RNase-free water instead of the recommended 22 µl RNase-free water. Barcoded fragment libraries were constructed using the Bovine Custom Any Deplete Ovation SoLo RNA-Seq System from NuGEN (San Carlos, CA, USA) following the manufacturer’s protocol.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
PP_5
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Data processing |
Sequence reads for each sample were mapped to the annotated bovine reference genome (UMD3.1.70) using CLC genomics workbench 12.0 (www.qiagenbioinformatics.com) Gene expression levels were assessed by counting the number of mapped reads for each Ensembl ID. Raw counts were processed with the edgeR package for the R software. Genes with low expression counts (less than 1 count per million) were filtered out before normalization. The between-samples normalization method applied was TMM (weighted trimmed mean of M-values). Genome_build: bosTau8 Supplementary_files_format_and_content: tab delimited text file containing raw read counts for each sample.
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Submission date |
May 09, 2019 |
Last update date |
Aug 31, 2019 |
Contact name |
Maria Belen Rabaglino |
E-mail(s) |
m.b.rabaglino@uu.nl
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Organization name |
Utrecht University
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Department |
Population Health Science
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Street address |
Yalelaan 7
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City |
Utrecht |
ZIP/Postal code |
3584 CL |
Country |
Netherlands |
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Platform ID |
GPL23055 |
Series (1) |
GSE130954 |
Molecular fingerprint of in vitro produced bovine embryos with high competence to establish pregnancy |
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Relations |
BioSample |
SAMN11609669 |
SRA |
SRX5811607 |
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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