Prior to fluorescence activated cell sorting (FACS), prim-staged embryos were manually deyolked by gently pipetting them up and down in ice-cold Ringer's Solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH7.2). Using a glass pipette coated with foetal bovine serum, deyolked embryos were transferred to a petri dish containing fresh ice-cold Ringer's Solution. The spinal cord region immediately above the yolk extension was dissected. For the CiA (V1) samples (collected from the Tg(pax2a:GFP) line), the pronephros region was removed to prevent contamination during FACS. The dissected tissue was then dissociated using the Worthington Papain Dissociation System (Worthington Biochemical Corporation, LK003150). Dissociated cells were resuspended in 0.5 ml Leibovitz's L-15 Medium (Thermo Fisher Scientific, 11415064) + 0.5% foetal bovine serum. All FAC-sorting was performed within four hours of commencing the spinal cord dissections. Cell suspensions were filtered to remove cellular aggregates using 35 µm Cell Strainer Cap Tubes (BD Falcon, 352235) and then FAC-sorted using a Beckman Coulter 3-laser MoFlo cytometer. Cells were sorted directly in to 100 µl of RNA extraction lysis buffer from the Ambion RNaqueous-Micro Total RNA Extraction kit (Thermo Fisher Scientific, AM1931) and immediately stored at -80oC.
Embryos were raised at 28.5oC until 10 hours post fertilisation (hpf), and then incubated at 32oC until they reached 27 hpf. Developmental stage was confirmed by prim-staging.
Total RNA was extracted from FAC-sorted cells using the Ambion RNaqueous-Micro Total RNA Extraction Kit (Thermo Fisher Scientific, AM1931), following the manufacturer's instructions. The optional DNase-treatment step was not performed. Extracted total RNA was eluted in 20 µl of nuclease-free water (two 10 µl elution volumes subsequently combined). RNA integrity was analyzed using the Agilent RNA 6000 Pico Kit (Agilent, 5067-1513) on the Agilent 2100 Bioanalyzer. Total RNA concentration was subsequently quantified using the Quant-IT RiboGreen RNA Assay Kit (Thermo Fisher Scientific, R11490) and a Mx3000P qPCR machine (Stratagene). A triplicate series containing 0, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5 and 10 ng of the ribosomal RNA standard provided in the kit was used to construct a standard curve of fluorescence versus RNA quantity from which the concentration of 0.5 µl aliquots of each experimental RNA sample was inferred.
As per the manufacturer's instructions, total RNA extracted from FAC-sorted cells was subjected to two rounds of amplification before labelling using the Affymetrix Two-Cycle Target Labeling and Control Reagents Kit (Affymetrix, 900494). As recommended by the manufacturer, Affymetrix GeneChip spike-in polyadenylated prokaryotic RNA controls were included as amplification controls.
The amplified RNA was hybridized to the Affymetrix GeneChip Zebrafish Genome Array according to the manufacturer's instructions. Affymetrix GeneChip Hybridization Control RNAs were always included.
Slides were scanned immediately after washing on the Affymetrix GeneChip Scanner 3000 7G, according to the manufacturer's instructions.
The scanned images were analyzed with Affymetrix Expression Console software and the following critical metrics determined: background, noise, probe cell intensity, scaling factor and the percent of probe sets called present or absent. Once satisfied that all of our hybridizations were successful and as consistent as could be expected, the data were subjected to an analysis pipeline incorporating background correction using the Robust Multi-chip Algorithm (RMA) and quantiles normalization. This was performed using the T-rex utility at the Gene Expression Profile Analysis Suite (GEPAS; www.gepas.org, Montagner et al., 2006 - now incorporated in to Babelomics 5; http://babelomics.bioinfo.cipf.es, Alonso et al., 2015). Subsequently in T-rex, a multi-class ANOVA test was performed to calculate differential expression. P-values were corrected for multiple testing using the Benjamini & Hochberg (BH) method.
RNA Profiling of FAC-Sorted Neurons From the Developing Zebrafish Spinal Cord.
Data table header descriptions
Normalization was performed using the default normalization tool for Affymetrix arrays provided in the T-rex utility at GEPAS. This utilizes the expresso and RMA functions from the Bioconductor Affy package (version 1.14.2), and also the GCRMA function from the Bioconductor GCRMA package (version 2.8.1) (http://www.bioconductor.org/).