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SRX377952: GSM1265769: 8-cell_replicate3; Bos taurus; RNA-Seq
1 ILLUMINA (Illumina Genome Analyzer IIx) run: 20.6M spots, 1.6G bases, 951.5Mb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: Fine mapping of genome activation in bovine embryos by RNA sequencing
show Abstracthide Abstract
During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the 4-cell, 8-cell, 16-cell and blastocyst stages. These were produced by in vitro fertilization of Bos t. taurus oocytes with sperm from a Bos t. indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 10^3 different genes were detected in the various developmental stages. EGA was analyzed by i) detection of embryonic transcripts which are not present in oocytes; ii) detection of transcripts from the paternal allele; and iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the 4-cell to the blastocyst stage. The largest proportion of gene activation, i.e. (i) 59%, (ii) 42%, and (iii) 58%, was found in 8-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the 4-cell stage identified categories related to translation, RNA processing and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and new insight into the timing of embryonic activation of specific genes. Overall design: RNA-Seq profiles from pools of 10 ooytes/embryos from bovine Bos t. taurus GV and MII oocytes and a cross-breed of Bos t. taurus x Bos t. indicus from 4-cell, 8-cell, 16-cell and blastocysts generated using Illumina GAIIx
Sample: 8-cell_replicate3
SAMN02403659 • SRS502758 • All experiments • All runs
Organism: Bos taurus
Library:
Instrument: Illumina Genome Analyzer IIx
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Frozen pools of ten oocytes or embryos were thawed and lysed in 10 µl of Lysis Buffer (Prelude kit from NuGEN, San Carlos USA). cDNA was generated and amplified with the ovation RNAseq v2 kit (NuGEN, San Carlos, USA). In brief 1 µl of the lysate was used for mixed random-/polyA-primed first-strand cDNA synthesis. After second strand synthesis the double-stranded cDNA was amplified by single primer isothermal amplification and the amplified cDNA was bead-purified (AmpureXP, Beckman-Coulter, Brea, USA) and fragmented by sonication (Bioruptor, Diagenode, Liege Belgium; 25 cycles 30 sec on/30 sec off). 500 ng of fragmented cDNA were used for preparation of Illumina-compatible sequencing libraries using the NuGEN Rapid library kit according to the manufacturer’s protocol. Adapter ligation was done with sample-specific barcodes. The resulting library was amplified (KAPA hifi polymerase, 8 cycles, 95°C 80 sec, 55°C 30 sec, 72°C 60 sec) and quantified on a Bioanalyzer 2100 (Agilent, Santa Clara, USA). Barcoded libraries were pooled at 10 nM concentration for multiplexed sequencing. Three replicates of each stage were sequenced on an Illumina GAIIx to a mean coverage of 20 Mio reads each. Sequencing runs were done in single-read mode with an 80 base read-length.
Experiment attributes:
GEO Accession: GSM1265769
Links:
External link:
Runs: 1 run, 20.6M spots, 1.6G bases, 951.5Mb
Run# of Spots# of BasesSizePublished
SRR103030820,598,5311.6G951.5Mb2014-02-10

ID:
539848

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