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| Status |
Public on Sep 01, 2014 |
| Title |
Rho v. Green E6 + 9DIV, Rho |
| Sample type |
SRA |
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| Source name |
Retinal explant culture
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| Organism |
Gallus gallus |
| Characteristics |
tissue harvest: embryonic day 6 (H&H Stage 26-28)
electroporated with: Rho::DsRed and Green::GFP
days in vitro: 9 DIV
cell type: Retinal explant culture
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| Treatment protocol |
Retinal explants were disociated using trypsin and trituration. For each experiment, two populations (DsRed+ and GFP+) were isolated from the same set of explants using FACS.
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| Growth protocol |
Eggs were maintained at 38°C in a rocking, humidified chamber for 6 days. Retinas were then harvested and electroporated with reporter constructs, and grown in culture as explants for either 9 or 15 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Rneasy kit, with on-column DNase treatment. cDNA was generated and amplified using the NuGen Ovation RNAseq kit.
Libraries were generated with Illumina adapters, and barcoded to allow all 6 samples for a single experiment (3 biological replicates for each of two cell populations) to be run on a single lane.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Sample 1
amplified cDNA (NuGen Ovation kit)
processed data file: rhogrn_early_cuffdiff_out.diff.txt
processed data file: rhogrn_early_edgeR_out.csv
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| Data processing |
Basecalls performed using Casava v1.7 (samples 1 and 2) or Casava v1.8 (samples 3-10)
Reads were aligned to galGal4 using Bowtie v0.12.7 and splice junctions called with TopHat v1.4.0
Differential expression was determined using cufflinks v1.3.0.
Differential expression was also determined by generating a counts table from the bowtie output, and then using R v.3.0.2 to implement edgeR.
Red, blue, and violet opsin are not fully represented in galGal4. To assess expression of these genes, reads were separately aligned to a set of 24 transcripts known to be expressed in photoreceptors, including all of the opsins, using Bowtie v.0.12.7 [The 24 transcripts are: Green Opsin (M92038.1), Red Opsin (NM_205440.1), Blue Opsin (NM_205517.1), Violet Opsin (NM_205438.1), RHO (NM_001030606.1), CRX (NM_204499.1), L-MAF (AF034570.1), NR2E3 (NM_204594.1), THRB (Z50188.1), RXRG (NM_205294.1), IRBP (AY994153.1), VSNL1 (NM_205255.1), GNAT2 (NM_204690.1), GNAT1 (NM_204691.1), PDE6C (NM_204986.1), PDE6H (NM_204408.1), CNGA1 (NM_205220.1), CNGA3 (NM_205221.1), ARR3 (NM_001097533.1), PDE6G (NM_204445.1), PDE6B (XM_424876.3), PDE6D (XM_422739.3), CRY1 (NM_204245.1), CNTFRA (U29245.1)]. Read counts for red, blue, and violet opsin were included in the edgeR analysis described above.
Genome_build: galGal4
Supplementary_files_format_and_content: cuffdiff output file containing tab-delimited differential gene expression data, and edgeR output as a comma separated values (csv) file
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| Submission date |
Jul 28, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Jennifer Enright |
| E-mail(s) |
enrightj@wusm.wustl.edu
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| Organization name |
Washington University School of Medicine
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| Street address |
660 S. Euclid, Box 8118
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| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL19005 |
| Series (1) |
| GSE59850 |
Transcriptome profiling of developing photoreceptor subtypes reveals candidate genes for avian photoreceptor diversification |
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| Relations |
| BioSample |
SAMN02943443 |
| SRA |
SRX664914 |
| 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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