|
| Status |
Public on Nov 30, 2011 |
| Title |
retina, Arc, P20 |
| Sample type |
RNA |
| |
|
| Source name |
Tg(Arc-EGFP)194Gsat/Mmcd
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Retinal Arc amacrine cell
batch: A and B
age (day): P20
genetic background: mixed FVB/N-Swiss Webster hybrid and C57BL/6J
line: Tg(Arc-EGFP)194Gsat/Mmcd
|
| 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. For exon arrays, the transcript cluster expression was calculated with the rma function using the command expr <- rma(read.celfiles[filenames, pkgname=pd.moex.1.0.st.v1]). Annotations for transcript clusters were downloaded from Affymetrix (MoEx-1_0-st-v1.na24.mm8.transcript.csv). The Arc-GFP time-course data was created in two batches: batch A contained samples P6-P16; batch B contained two non-contiguous sets of samples, P1-P6 and P17-P43. Correlation analysis revealed extensive batch effects between the samples. Because time-course batch A was surrounded on both sides by batch B, we shifted (in log2 space) the expression values for each transcript in batch A so that the average expression in batch A corresponded to the average expression in batch B. That is why we have in the matrix file only 1 value per Data point for Arc
MoEx-1_0-st-v1.r2_core.pgf
MoEx-1_0-st-v1.r2.dt1.mm8.core.mps
|
| |
|
| Submission date |
Oct 19, 2011 |
| Last update date |
Nov 30, 2011 |
| Contact name |
Sandra Siegert |
| E-mail(s) |
[email protected]
|
| Organization name |
Friedrich Miescher Institute
|
| Street address |
Maulbeerstrasse 66
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL6096 |
| Series (2) |
| GSE33088 |
Developmental time-course of adult cell-type-specific retina genes of amacrine cells |
| GSE33089 |
Retina cells |
|
