|
| Status |
Public on Nov 30, 2011 |
| Title |
retina, adult, ChAT 0, cone 200, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Tg(Chrnb4-EGFP)1Gsat
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Retinal Cone photoreceptor
genetic background: mixed FVB/N-Swiss Webster hybrid and C57BL/6J
cell mixture: ChAT 0, cone 200
line: Tg(Chrnb4-EGFP)1Gsat
developmental stage: adult
|
| Growth protocol |
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.
|
| |
|
| Submission date |
Oct 19, 2011 |
| Last update date |
Nov 30, 2011 |
| Contact name |
Sandra Siegert |
| E-mail(s) |
ssiegert@mit.edu
|
| Organization name |
Friedrich Miescher Institute
|
| Street address |
Maulbeerstrasse 66
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL6246 |
| Series (2) |
| GSE33076 |
Linearity of amplification between gene expression values and the amounts of RNA in a retina cell group |
| GSE33089 |
Retina cells |
|
