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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 20, 2013 |
| Title |
germring.RNA-seq |
| Sample type |
SRA |
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| Source name |
embryo
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| Organism |
Danio rerio |
| Characteristics |
devolopmetal stage: germ ring
stain: Tubingen
tissue: embryo
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| Extracted molecule |
total RNA |
| Extraction protocol |
Zebrafish embryos were chilled on ice, washed by pre-chilled PBS buffer three times to avoid the contamination and homogenized in 10 vol buffer N (10 mM HEPES, pH 7.5, 250 mM sucrose, 50 mMNaCl, 5 mM MgCl2, 10 µg/ml cytochalasin B, 1 mMdithiothreitol and protease inhibitors) with ten strokes of a loose fitting pestle in a glass homogenizer, and lysates were decanted for 20 min on ice. Then lysates were filtered with 70µm cell strainer（BD Biosciences) and centrifuged through 1 M sucrose at 1000 g for 30 min. Pelleted nuclei from embryos were subjected to genomic DNA extraction by standard phenol precipitation method. Total RNA was extracted from homogenized whole embryos using Trizol (Invitrogen) according to manufacturer's instructions.
The RNA-seq libraries were constructed as per the TruSeq RNA Sample Preparation Guide (Illumina). Briefly, mRNA was purified using poly-T oligo-attached magnetic beads and fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were then reverse transcribed into first strand cDNA, followed by second strand cDNA synthesis to generate double-stranded (ds) cDNA. After end repair and dA tailing, multiple indexing adapters were ligated to the ends of the double strand cDNA. The ligation products were size selected to contain 350-450bp fragments and purified. Finally, DNA fragments that have adapters on both ends were enriched using PCR to create the final sequencing library and sequenced by Illumina HiSeq 2000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality
FASTQ sequence files were aligned to the Zebrafish reference (Zv9) using BWA Version: 0.6.1-r104, retaining only unique, non-duplicate genomic matches with no more than 2 mismatches within the first 32bp.
Using uniquely mapped reads, read counts per exon were obtained, followed by summarizing the read counts for all exons within a gene for all genes in UCSC Zv9 refFlat table. For each sample, RPKM was computed as the number of reads which map per kilobase of exon model per million mapped reads for each gene.
As there are global shifts in the levels of mRNA during embryonic development(Aanes et al., 2011). TMM (Trimmed Mean of M values)(Robinson and Oshlack, 2010) method implemented in R package edgeR was used to normalize the global shifts in gene expression, resulting in a shifting factor (TMM factor) for each sample. Briefly, the relative abundance of each transcript was estimated as the number of reads for transcript i divided by the total number of reads in sample j. Eij=Rij/Rj, where Eij is the relative abundance of transcript i in sample j, Rij is the number of reads for gene i in sample j, and Rj is the total number of reads in sample j. The average Rj for all samples were summed to generate a common library size: Xavg = sum(R1~Rn)/n. A pseudo library size were calculated by multiplying Xavg with the TMM factor (Xj = Xavg*Zj), where Zj was TMM factor computed as described above. These pseudo read libraries were reassigned to each gene based on previously estimated Eij value to get the normalized dataset (normEij = Eij*Xj).
Genome_build: Zv9/danRer7
Supplementary_files_format_and_content: tab-delimited text files include reads count of all exons within a gene for all genes, which is TMM-normalized among sperm ,egg , 1kcell and germring samples.
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| Submission date |
Feb 20, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Lan Jiang |
| E-mail(s) |
lan_jiang@med.harvard.edu
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| Organization name |
Beijing Institute of Genomics
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| Lab |
Liu Jiang
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| Street address |
NO.1 Beichen West Road, Chaoyang District
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| City |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
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| Platform ID |
GPL14875 |
| Series (1) |
| GSE44075 |
Sperm but not oocyte DNA methylome is inherited by zebrafish early embyros |
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| Relations |
| SRA |
SRX243927 |
| BioSample |
SAMN01924086 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1085062_germring.normalized.reads.count.TXT.gz |
112.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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