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| Status |
Public on Feb 06, 2014 |
| Title |
Seq14 |
| Sample type |
SRA |
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| Source name |
sorted cardiomyocytes
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| Organism |
Danio rerio |
| Characteristics |
developmental stage: 24hpf
transgenic line: tg(myl7::gfp)
experimental group: Gata4 morphant
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| Treatment protocol |
Embryos were injected with morpholino before the 4-cell stage of development. 2nl of a 0.7mM concentration of Gata4 morpholino (5'-TCCACAGGTGAGCGATTATTGCTTC-3') were injected per individual embryo. At 24 hours post fertilization (hpf), batches of approximately 200 embryos were pooled into 1.5 ml tube. Embryos were dissociated by manual agitation with a pellet pestle (Fisher) and trypsinized with pre-heated TrypLE (Life Technologies) at 32C for 15 min on a rotator. Trypsinized samples were pipetted through a 35um cell strainer into a 5ml tube, trypsin inhibited by addition of 4ml FACS buffer (L-15 medium supplemented with 1% heat inactivated FCS, 0.8mM CaCl2, 50U/ml penicillin, and 0.05 mg/ml streptomycin) followed by addition of FCS to 7.5% final concentration. Cells were pelleted at 300 RCF for 5 min and then washed with FACS buffer. Dissociated embryonic cells were resuspended at 7.5x106 cells/ml in FACS buffer. FACS was performed on a Vantage cell sorter (BD) into Trizol LS (Life Technologies), and stored at -80C until RNA isolation.
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| Growth protocol |
Embryos were grown in 1x E3 buffer for 24 hours at 28.5 degrees Celsius in petri dishes. 25 ml of E3 buffer was used per 50-60 embryos in each dish.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated by Trizol except that after addition of 1.5 volumes of 100% ethanol to the aqueous phase, the solution was transferred to an RNeasy minElute column (Qiagen). On-column DNase digestion, subsequent washing, and RNA elution was performed according to the Qiagen's recommended protocol for RNeasy micro kit.
100ng of total RNA was used to prepare the libraries. Libraries were prepared for RNA sequencing using the mRNA-Seq (seq1, seq5, and seq11) or TruSeq Kit (seq13, seq14, and seq15) according to the Illumina's recommended protocol.
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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, demultiplexing, and filtering of reads was performed using Casava 1.7.0.
Read alignment was performed with the BWA alignment algorithm with native goby support using gobyweb, version: development (20110921150240) with the following parameters: Ambiguity threshold = 1, Max Number Gap Opens = 1, Max Number Gap Extensions = -1.
Differential expression was generated with the DESEQ package with native goby support using gobyweb, version: development (20110921150240) with the following parameters: q-value threshold = 1.0, weight adjustment = none, Gene counts box checked.
Data were filtered according to geneID's demonstrating a p-adjusted < 0.1, a log2 fold change > 1 for WT/G4, and Average RPKM in the comparison group > 1.
Genome_build: Zv9.61
Supplementary_files_format_and_content: Wig files were generated using gobyweb, version: development(20110921150240). Additional supplementary .xlsx file containing RPKM, counts, and statistical values was exported from gobyweb and processed in excel.
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| Submission date |
Feb 11, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Gabriel Rosenfeld |
| E-mail(s) |
gar2007@med.cornell.edu
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| Organization name |
Weill Cornell
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| Department |
Cell and Developmental Biology
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| Lab |
Todd Evans Lab
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| Street address |
1300 York Ave. Rm. LC711
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL14875 |
| Series (1) |
| GSE44233 |
Comparison of cardiomyocyte transcripts after knockdown of Gata4 in zebrafish embryos |
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| Relations |
| SRA |
SRX233124 |
| BioSample |
SAMN01919349 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1081112_seq14-all.wig.gz |
17.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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