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| Status |
Public on Jun 29, 2012 |
| Title |
2DPFETOH rep3 |
| Sample type |
SRA |
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| Source name |
Zebrafish embryos/larvae
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| Organism |
Danio rerio |
| Characteristics |
genotype: Segrest wild type
age: 2 days post fertilization
treatment: ethanol
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| Treatment protocol |
Zebrafish embryos were obtained from mating of Segrest wild-type (SWT) parents under controlled barrier conditions, in the Mayo Clinic Zebrafish Core Facility, in Instant Ocean media . Zebrafish embryos (25-30) were placed in 20 mL embryo medium (pH 7.2) containing 1-phenyl-2-thiourea (PTU) (0.003% (w/v) and were maintained at 28-30 oC. At 24 hpf (1 day post fertilization, dpf), 10 microliters of 1a,25(OH)2D3 in ethanol was added to embryos maintained in 20 mL fresh embryo medium with PTU. The final concentration of 1a,25(OH)2D3 was 300 pM. Control zebrafish were treated with 10 microliters ethanol alone (vehicle controls). The medium containing either 300 pM 1a,25(OH)2D3 or vehicle was changed every 24 h . In experiment 1, at 2, 4, 6 and 7 dpf embryos/larvae were removed and immediately frozen at -80 0C for later RNA preparations. 25-30 embryos per set were used for preparation on RNA. At the same times, 7-12 embryos were fixed in 4% paraformaldehyde in 0.75 X Dulbecco’s phosphate buffered saline (DPBS). In experiment 2, 6 dpf larvae were treated with 1a,25(OH)2D3 (300 pM) or vehicle for 24 h. RNA was prepared from three sets of larvae.
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| Growth protocol |
Zebrafish embryos were obtained from mating of Segrest wild-type (SWT) parents under controlled barrier conditions, in the Mayo Clinic Zebrafish Core Facility, in Instant Ocean media . Zebrafish embryos (25-30) were placed in 20 mL embryo medium (pH 7.2) containing 1-phenyl-2-thiourea (PTU) (0.003% (w/v) and were maintained at 28-30 oC. At 24 hpf (1 day post fertilization, dpf), 10 microliters of 1a,25(OH)2D3 in ethanol was added to embryos maintained in 20 mL fresh embryo medium with PTU. The final concentration of 1a,25(OH)2D3 was 300 pM. Control zebrafish were treated with 10 microliters ethanol alone (vehicle controls). The medium containing either 300 pM 1a,25(OH)2D3 or vehicle was changed every 24 h . In experiment 1, at 2, 4, 6 and 7 dpf embryos/larvae were removed and immediately frozen at -80 0C for later RNA preparations. 25-30 embryos per set were used for preparation on RNA. At the same times, 7-12 embryos were fixed in 4% paraformaldehyde in 0.75 X Dulbecco’s phosphate buffered saline (DPBS). In experiment 2, 6 dpf larvae were treated with 1a,25(OH)2D3 (300 pM) or vehicle for 24 h. RNA was prepared from three sets of larvae.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA preparation for RNA-seq and QPCR: RNA was prepared using RNA/protein spin columns (Clontech, Mountain View, CA). Lysis solution was added to 25-30 frozen embryos and embryos were lysed by passage of tissue successively through 21 and 27 gauge needles. Individual embryo lysates were applied to RNA spin columns for purification. Preparation of libraries: mRNA-seq libraries are prepared using the mRNA v1 sample prep kit following the manufacturer’s protocol (Illumina). Poly-A containing mRNA is purified using poly-T oligo attached magnetic beads. The purified mRNA is fragmented using divalent cations at 95°C for 5 minutes and ethanol precipitated. The RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Second strand cDNA synthesis is performed using DNA polymerase I and RNase H. The cDNA is purified using Qiaquick PCR columns from Qiagen. The ends are repaired and phosphorylated using Klenow, T4 polymerase, and T4 polynucleotide kinase, after which an “A” base is added to the 3’ ends of double-stranded DNA using Klenow exo- (3’ to 5’ exo minus). Paired end DNA adaptors (Illumina) with a single “T” base overhang at the 3’ end are ligated and the resulting constructs are separated on a 2% agarose gel. DNA fragments of approximately 250-300 bp are excised from the gel using GeneCatcher tips and purified using Qiagen Gel Extraction Kits. The adapter-modified DNA fragments are enriched by 15 cycles of PCR using primers PE 1.0 and PE 2.0 (Illumina). The concentration and size distribution of the libraries is determined on an Agilent Bioanalyzer DNA 1000 chip. Libraries are loaded onto paired end flow cells at concentrations of 6 pM to generate cluster densities of 400,000/mm2 following Illumina’s standard protocol using the Illumina cBot and cBot Paired end cluster kit version 2. The flow cells are sequenced as 51 X 2 paired end reads on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 1 or version 3 and SCS version 1.1.37.0 or HCS version 1.4.8 data collection software, respectively. Base-calling is performed using Illumina’s RTA version 1.7.45.0.
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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 |
Basecalls performed using CASAVA version 1.4
mRNA-seq reads were aligned to the ZV9 assembly using Bowite 0.12.7 with the following configurations…
Gene and exon level count data were generated by HTSeq 0.5.3p3
Genome_build: ZV9
Supplementary_files_format_and_content: tab-delimited text files include raw count numbers at gene and exon level for each Sample ...
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| Submission date |
Jun 27, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuji Zhang |
| E-mail(s) |
yuzhang@som.umaryland.edu
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| Phone |
410-706-8523
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| Organization name |
University of Maryland
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| Street address |
660 W. Redwood Street
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21201 |
| Country |
USA |
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| Platform ID |
GPL14875 |
| Series (1) |
| GSE38575 |
Whole Transcriptome RNA Sequencing Detects Multiple 1a,25-Dihydroxyvitamin D3-Sensitive Metabolic Pathways in Zebrafish Embryos |
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| Relations |
| SRA |
SRX158607 |
| BioSample |
SAMN01086486 |
| 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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