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Sample GSM819276 Query DataSets for GSM819276
Status Public on Nov 30, 2011
Title retina, adult, b2 2nd, biological rep1
Sample type RNA
 
Source name b2-Cre × Rosa26-LSL-RFP
Organism Mus musculus
Characteristics cell type: Retinal Rod photoreceptor
batch: 2
genetic background: mixed FVB/N-Swiss Webster hybrid and C57BL/6J
line: b2-Cre × Rosa26-LSL-RFP
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
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.
 
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)
GSE33085 Transcriptome analysis of adult retina cell types.
GSE33089 Retina cells

Data table header descriptions
ID_REF
VALUE Linearized expression value, rod-cone contamination normalized (as described in data processing)

Data table
ID_REF VALUE
10344620 4.932753343
10344624 8.231579891
10344633 85.96753807
10344637 162.0948284
10344653 5.426588657
10344658 15.89304438
10344674 5.725776962
10344679 10.67673873
10344707 25.00575427
10344713 33.2138638
10344719 16.90035362
10344723 111.6236216
10344725 7.764138946
10344741 7.309943551
10344743 5.148127301
10344750 7.87528868
10344772 5.192538053
10344789 7.437135364
10344837 3.885963493
10344879 3.405234199

Total number of rows: 22347

Table truncated, full table size 455 Kbytes.




Supplementary file Size Download File type/resource
GSM819276_ro20100228mg10_04_b2-CrexRosa26-LSL-RFP_20.01.10_1_385_MoGene-1_0-st-v1_.CEL.gz 3.2 Mb (ftp)(http) CEL
Processed data included within Sample table

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