|
| Status |
Public on Sep 08, 2014 |
| Title |
Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing |
| Organism |
Danio rerio |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
We describe a method for isolating RNA suitable for high-throughput RNA sequencing (RNA-seq) from small numbers of fluorescently labeled cells isolated from live zebrafish (Danio rerio) embryos without using costly, commercially available columns. This method ensures high cell viability after dissociation and suspension of cells and gives a very high yield of intact RNA. We demonstrate the utility of our new protocol by isolating RNA from fluorescence activated cell sorted (FAC sorted) pineal complex neurons in wild-type and tbx2b knockdown embryos at 24 hours post fertilization. Tbx2b is a transcription factor required for pineal complex formation. We describe a bioinformatics pipeline used to analyze differential expression following high-throughput sequencing and demonstrate the validity of our results using in situ hybridization of differentially expressed transcripts. This protocol brings modern transcriptome analysis to the study of small cell populations in zebrafish.
|
| |
|
| Overall design |
Differential expression analysis of mRNA levels in a single time-point (24 hpf) between wild-type and Tbx2b deficient FAC sorted pineal complex cells
|
| |
|
| Contributor(s) |
Khuansuwan S, Gamse JT |
| Citation(s) |
25173875 |
| Submission date |
Sep 08, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Sataree Khuansuwan |
| E-mail(s) |
sataree.khuansuwan@gmail.com
|
| Organization name |
Vanderbilt University
|
| Department |
Biological Sciences
|
| Lab |
Gamse Laboratory
|
| Street address |
465 21st Avenue South
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL14875 |
Illumina HiSeq 2000 (Danio rerio) |
|
| Samples (8)
|
|
| Relations |
| BioProject |
PRJNA260501 |
| SRA |
SRP046327 |
Less...
More...