Experiment SRX271958 Download

TitleGSM1129620: dm_77h_28_399s62; Danio rerio; RNA-Seq
Design descriptionno content
Referencesno content
OrganismsDanio rerio
SubmitterGene Expression Omnibus
InstrumentIllumina HiSeq 2000
Library nameno content
Library strategyRNA-Seq
Library selectioncDNA
Library layoutPAIRED
Library construction protocolThis study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor (mitfavc7). This mutant facilitated the collection of RPE, as mitfa is not required for RPE development in zebrafish. Melanocytes, iridophores, and RPE develop normally at 25°C in mitfavc7. When held at 32°C, the neural crest-derived melanocytes do not develop, but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine, rinsed with Ca-, Mg- DPBS (Sigma, D8537), and immersed in 100mL TrypLE Express (Invitrogen, 12604039) per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15-20 minutes, followed by trituration with a Pasteur pipette to remove eyes from larva. After separation of eyes and larva, each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1-1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10-20 times, and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor (Eppendorf 5810 R) at 500 relative centrifugal force (rcf) for 5 minutes at 4°C, then resuspended in 1mL cold isotonic Percoll (Sigma, P1644) by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum (FCS), and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10,000rcf for 15 minutes at 4°C in a fixed angle micro-centrifuge (Eppendorf 5415 R). Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation, overlying Percoll was aspirated, leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS, and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence-Activated Cell Sorting (FACS). Following resuspension in 500µL cold DPBS with 2% FCS, the enriched cell populations were screened through a 30uM cell filter (Partec, 04-0042-2316). Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive (DD) frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter, selecting against the smallest particles (~1µm or less). Cells were sorted based on detection using 510-530nm and 575-595nm filters, corresponding to FL1 and FL2 in Figure 1D, respectively. When excited by the 488 nm laser, the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser, and cluster at the lower left of the FACS plot. Cells were collected into ice-cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit (Invitrogen) was used per manufacturer's instructions. Following mRNA elution from the Dynabeads, first strand cDNA synthesis was performed using MMLV reverse transcriptase (Clontech) using an anchored polyT primer tailed with a universal primer sequence (See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.) A universal primer sequence was also added to the 3' end of the first strand by template switching, allowing for PCR-amplification of the resultant cDNA [43,44]. Following PCR amplification using the high fidelity polymerase LA Taq (TaKaRa, PCR cycle: 95C for 1 minute, followed by 20 cycles of 98C for 25 seconds, 60C for 1 minute, 68C for 20 minutes), cDNA was digested with AluI and RsaI restriction enzymes (NEB). Blunt-end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end-repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites, we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed, performed by the Genome Technology Access Center (GTAC) at Washington University in St. Louis (http://gtac.wustl.edu). In brief, a single A was added to the 3’ end of each strand, Y-adapters ligated, and library enrichment PCR performed, followed by gel extraction size-selection for fragments ranging from 200-400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.
Related objects
LinksGEO Sample, Unknown DB(gds)
GEO Accession GSM1129620