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| Status |
Public on Feb 08, 2018 |
| Title |
GFPneg5_2 |
| Sample type |
SRA |
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| Source name |
Zebrafish retinal cells, not rods
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| Organism |
Danio rerio |
| Characteristics |
cell type: non-rod retinal cells
genotype/variation: XOPS:eGFP
cell type: GFP-; FACS-sorted non-rods from retina
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| Treatment protocol |
Whole retinas were dissociated into cell suspensions by incubating with 0.225% trypsin (Fisher ThermoScientific) and 0.001% papain (Sigma) for 10 minutes at 37°C. Dissociation was stopped by the addition of fetal bovine serum (10% v/v final concentration). Suspended cells were pelleted and incubated with DNAseI at room temperature for 15 min. Cells were pelleted and resuspended in 100 µL phosphate-buffered (pH 6.5) saline (PBS) and immediately FACS-sorted. GFP+ vs. GFP- retinal cells were sorted using a FACSAria flow cytometer, using the 488 nm laser and FITC fluorescence filter, and the 70 µm nozzle. Some cells were collected for fluorescence microscopy, or for post-sort FACS analysis. For RNA-seq or qPCR, GFP+ and GFP- cells were collected separately in a final volume of 100 µL of the FACS sheath fluid, and RNA was immediately extracted.
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| Growth protocol |
Zebrafish (XOPS:eGFP transgenic line) were on a 14:10 light:dark cycle in recirculating, monitored system water, housed and propagated according to The Zebrafish Book (Westerfield).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from tissue samples using the NucleoSpin® RNA kit (Macherey-Nagel) using the manufacturer’s protocol, quantified and quality-checked on a Nanodrop spectrophotometer. Both quantity and quality were assessed by using an Agilent 2100 Bioanalyzer. All samples used for RNA-seq had an RNA integrity number (RIN) > 8.0, and experimental design was such that GFP+ cells, or GFP- cells derived from the two retinas of a single fish constituted each RNA sample as a biological replicate. At least 5 ng of RNA was available per sample, and provided to the University of Idaho’s Institute for Bioinformatics and Evolutionary Studies (IBEST) Genomics Core for RNA amplification and the generation of cDNA.
Quality and quantity of cDNA were verified by Bioanalyzer. All sample preparation was achieved with Ovation® RNA-Seq System V2 (NuGEN), and sequencing performed on an Illumina (San Diego, CA) MiSeq. Four replicates (from four different fish) were sequenced (Fig. 1A). Mapping percentages ranged from 97.0% to 97.4% per sample, and sequencing depth ranged from 2,781,516 to 3,480,515 reads per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
GFPneg5_2
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| Data processing |
Quality trimmed with Sickle (https://github.com/najoshi/sickle)
Paired reads were overlapped with FLASH (https://ccb.jhu.edu/software/FLASH/)
Overlapped reads were against the Danio rerio reference using BWA (http://bio-bwa.sourceforge.net/)
BAMs were sorted using SAMtools (http://samtools.sourceforge.net/)
Reads were counted by feature using htseq-count (http://www-huber.embl.de/HTSeq/doc/count.html)
Counts were analyzed and differentially expressed genes were identified with R and edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html)
Genome_build: Zv9.75
Supplementary_files_format_and_content: Read count data from HTSeq; EdgeR analysis results with annotation
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| Submission date |
Jun 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Deborah Lynn Stenkamp |
| E-mail(s) |
dstenkam@uidaho.edu
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| Phone |
208-885-8963
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| Organization name |
University of Idaho
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| Department |
Biological Sciences
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| Street address |
875 Perimeter Drive, MS 3051
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| City |
Moscow |
| State/province |
Idaho |
| ZIP/Postal code |
83844-3051 |
| Country |
USA |
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| Platform ID |
GPL21930 |
| Series (1) |
| GSE100062 |
Transcripts within Rod Photoreceptors of the Zebrafish Retina |
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| Relations |
| BioSample |
SAMN07246698 |
| SRA |
SRX2921961 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2670727_GFPneg5_2_sn.count.txt.gz |
116.7 Kb |
(ftp)(http) |
TXT |
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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