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Sample GSM1265769

Query DataSets for GSM1265769

Status

Public on Feb 09, 2014

Title

8-cell_replicate3

Sample type

SRA

Source name

embryos

Organism

Bos taurus

Characteristics

Stage: 8-cell
breed: German Simmental x Brahman

Growth protocol

IVP of bovine embryos was done as described previously . Briefly, cumulus-oocyte complexes (COCs) were aspirated from German Simmental ovaries obtained in a local abattoir and washed twice in preincubated (39°C, 5% CO2 in air) Tissue Culture Medium 199 (TCM 199; Invitrogen) supplemented with l-glutamine (100 mg/L), NaHCO3 (3 g/L), Hepes (1400 mg/L), sodium pyruvate (250 mg/L), l-lactic-Ca-salt (600 mg/L), gentamicin (55 mg/L), and 10% (v/v) estrous cow serum (ECS). For in vitro maturation (IVM) COCs were washed in TCM 199 supplemented with 0.01 U/ml of bovine FSH and bovine LH (both Sioux Biochem), transferred to four-well plates (Nunc), and matured in 400 μl of this medium for 20–24 h at 39°C in an atmosphere of 5% CO2 in humidified air. For in vitro fertilization (IVF) the matured COCs were transferred to 400 μl of Tyrode albumin lactate pyruvate (TALP) medium containing 2.2 mg/ml of sodium pyruvate, 10 μg/ml of heparin sodium salt, and 6 mg/ml of BSA, after three washes in the same medium. Then, the COCs and spermatozoa (artificial insemination straws from a commercially available Zebu bull, subjected to a 90 min swim-up after thawing) were co-incubated for 18 h at 39°C in an atmosphere of 5% CO2 in humidified air. Presumptive zygotes were mechanically denuded by vortexing and gentle pipetting; washed three times in synthetic oviduct fluid (SOF) culture medium enriched with 5% ECS, 20 μl/ml of 100× basal medium (Invitrogen), and 10 μl/ml of 100× minimum essential medium (Invitrogen). Batches of app. 20 embryos were transferred to 400-μl droplets of medium covered with mineral oil and cultured until reaching the respective stages. The culture atmosphere was 5% CO2, 5% O2, and 90% N2 at 39°C and maximum humidity. Pools of ten embryos were picked after visual inspection and snap frozen in liquid nitrogen after washing in PBS. Stages collected for sequencing were: denuded oocytes before and after maturation and embryos at the 4-cell, 8-cell, 16-cell, and blastocyst stages.

Extracted molecule

total RNA

Extraction 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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

pools of 10 embryos

Data processing

CASAVA 1.6 software was used for basecalling.
Adaptor sequences and the first 5 bases were removed. Additionally, reads were trimmed from the 3'- and 5'-end with a quality cutoff of 20. Reads below a length of 30 were discarded.
Filtered reads were mapped with Tophat2 (v.2.0.3) to the bovine UMD3.1 reference genome with default parameters.
The number of reads falling into exons of genes were counted using HTSeq-count. Read counts were normalized by library size and by a variance stabilizing transformation with DESeq and differential abundances were calculated with an adj. p-value < 0.05.
Genome_build: UMD 3.1
Supplementary_files_format_and_content: tab-delimited text file include normalized values for each gene and sample.

Submission date

Nov 15, 2013

Last update date

May 15, 2019

Contact name

Alexander Graf

E-mail(s)

graf@genzentrum.lmu.de

Organization name

Ludwig-Maximilians-Universität München

Department

Gene Center Munich