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| Status |
Public on Mar 29, 2019 |
| Title |
DKK1_R |
| Sample type |
SRA |
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| Source name |
bovine embryo
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| Organism |
Bos taurus |
| Characteristics |
age: day 15 of development
length (mm): 50
developmental stage: embryo
treatment: 100 ng/ml DKK1
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| Treatment protocol |
Culture medium was supplemented with either 0 or 100 ng/ml human recombinant DKK1 on day 5 of culture. Embryos were transferred to recipient on day 7 and harvested on day 15.
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| Growth protocol |
Embryos were produced in vitro from oocytes from abattoir and sex-sorted semen, culture for 7 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from each embryo using the Qiagen RNeasy Mini kit (Qiagen, Valenciam, CA, USA) following manufacturer instructions, including DNase treatment. 25 ng of total RNA was used for mRNA isolation with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs).
RNA library construction was performed at the Interdisciplinary Center for Biotechnology Research (ICBR) Gene Expression & Genotyping Core, University of Florida. RNA libraries were constructed with NEBNext® Ultra™ Directional RNA Library Prep Kit (New England Biolabs) according to the manufacturer's user guide. Briefly, RNA was fragmented followed by first strand cDNA synthesis using reverse transcriptase and oligo-dT primers. Synthesis of double strand cDNA was performed, followed by end-repair and adaptor ligation. At this point, Illumina adaptors were ligated to the sample. The library was enriched (each library had a unique barcode) by 10 cycles of amplification, and purified by Agencourt® AMPure beads (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Finally, equimolar amounts of 20 individual libraries were pooled and sequenced by Illumina HiSeq 3000 2X100 cycles run for total of 3 runs (Illumina Inc., San Diego, CA, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Raw paired-end reads generated from the Illumina HiSeq 3000 sequencing platform were cleaned up with the Cutadapt program. The partial adaptors and potential sequencing errors introduced during sequencing or library preparation were trimmed off and reads with a quality and read length <40 bases were excluded.
All processed paired-end reads of each sample were individually mapped to the reference sequences using bowtie2 (v. 2.2.3) with a ‘3 mismatches within a read’ allowance.
The samtools and scripts developed in house at the ICBR were used to detect the potential PCR duplicates and to choose uniquely mapped reads for gene expression analysis.
Gene expression levels were assessed by counting the number of mapped reads for each gene symbol.
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 |
Feb 18, 2019 |
| Last update date |
Mar 30, 2019 |
| Contact name |
Maria Belen Rabaglino |
| E-mail(s) |
[email protected]
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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 |
GPL21659 |
| Series (1) |
| GSE126680 |
Effect of DKK1 on embryo elongation |
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| Relations |
| BioSample |
SAMN10962206 |
| SRA |
SRX5387676 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3611160_R_count.txt.gz |
56.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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