Mice obtained from Mutant Mouse Regional Resource Centers (MMRRC) were FVB/N-Swiss Webster hybrids that are known to harbor a recessive mutation causing photoreceptor degeneration. These were backcrossed into the C57BL/6J background (RCC) and the F2 generation was used for transcriptome analysis. All other mouse lines had already been in the C57BL/6J background for several previous generations.
Extracted molecule
total RNA
Extraction protocol
Retinal cells were dissociated using a modified Papain-dissociation protocol of Morrow et al. with a process time of only 15 min. Cells from the retina were sorted. Cell sorting was performed using a MoFlo (Cytomation, Fort Collins, USA) with HQ515/30 (GFP) and HQ616/26 (RFP) bandpass filters. After recording several thousand events the fluorescence gate was set using Summit V 4.3.01 Build 2449 software and a population of events selected. Size and granularity of this population were determined using the forward and side scatter values, respectively, and cell debris discarded by setting a second gate. Finally, after determination of the duration of the events (pulse-width), a third gate was selected that served to exclude cell doublets or clumps. Using these three gates, cells were sorted in single-cell drop mode. A total of 200 cells were sorted at room temperature (RT) into a low-binding tube (Eppendorf) containing 60 µl cell lysis buffer (TRI reagent, Sigma-Aldrich).
Label
biotin
Label protocol
We used transgenic mouse lines that showed fluorescent-protein expression (RFP or GFP) in distinct cell types within the retina.
Hybridization protocol
Hybridization was performed with 5 μg biotinylated target, which was incubated with the GeneChip Gene 1.0 ST array (Affymetrix) at 45°C for 16 h. GeneChip Mouse Exon 1.0 ST array was used for the developmental study. Following hybridization, non-specifically bound nucleotides were removed by washing and the specifically bound target was detected using the GeneChip Hybridization, Wash and Stain kit, and the GeneChip Fluidics Station 450 (Affymetrix).
Scan protocol
The arrays were scanned using a GeneChip Scanner 3000 7G (Affymetrix) and CEL files acquired using a GeneChip Command Console Software (Affymetrix).
Description
GFP
Data processing
Data analysis of gene arrays was done in R (version 2.11.1) using bioconductor as well as in Mathematica (Wolfram Research). For gene arrays, after obtaining the CEL files for each gene array, the files were normalized using the rma function in the oligo package. The resulting text file for gene arrays contained the normalized expression values for 35’556 probes at log2 scale. Mouse gene annotation was downloaded from Affymetrix (MoGene-1_0-st- v1.na30.1.mm9.transcript.txt). Probes were only analyzed when a gene entry was annotated and duplicated annotated genes were removed. All genes are referred to by their NCBI gene symbols. Rod mixtures. In a first pre-screen of the gene expression data, expression of known rod-specific genes was recorded in the transcriptomes of almost all cell groups, despite a lack of GFP- or RFP-labeling in rod photoreceptors in these mouse lines. To eliminate this rod contamination, we used the separation method described above treating rods as type 1 cells. Note that despite a priori knowledge of several cell type-specific genes in other groups, we did not find these genes expressed in “incorrect” cell groups, suggesting that contamination was only by rods. For example, we did not find cone opsin (Opn1mw, Opn1sw) or melanopsin (Opn4) expression in cell groups in which the corresponding retinas had no cone or melanopsin labeling, respectively. Cone mixtures. In the mouse lines Lhx4, Chrna3, and Ier5 both cones and other cell types were labeled. We separated these mixtures into cone (Type 1) and other cell type (Type 2) components by the procedure described above. We used the predicted transcriptome of the Type 2 component in our analysis.
