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Sample GSM819157 Query DataSets for GSM819157
Status Public on Nov 30, 2011
Title retina, adult, ChAT 43, cone 157, biological rep3
Sample type RNA
 
Source name ChAT-Cre × Rosa26-LSL-RFP : Chrnb4-GFP
Organism Mus musculus
Characteristics cell type: Retinal Starburst amacrine cell and cone photoreceptor mixture
genetic background: mixed FVB/N-Swiss Webster hybrid and C57BL/6J
cell mixture: ChAT 43, cone 157
line: ChAT-Cre × Rosa26-LSL-RFP : Chrnb4-GFP
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 RFP/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

Data table header descriptions
ID_REF
VALUE log2 GC-RMA

Data table
ID_REF VALUE
10344620 2.353344259
10344624 2.806448839
10344633 5.852706036
10344637 6.398792956
10344653 2.110206463
10344658 3.815441424
10344674 2.94966208
10344679 3.07419766
10344707 4.709180748
10344713 3.423477763
10344719 3.632699911
10344723 6.188208698
10344725 2.285246249
10344741 3.344534137
10344743 2.136574167
10344750 2.219974536
10344772 2.173933746
10344789 2.465554094
10344837 2.292765913
10344879 2.360192257

Total number of rows: 22347

Table truncated, full table size 455 Kbytes.




Supplementary file Size Download File type/resource
GSM819157_ro20100806mg10_24_x1_432_MoGene-1_0-st-v1_.CEL.gz 3.4 Mb (ftp)(http) CEL
Processed data included within Sample table

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