|
| Status |
Public on Apr 10, 2013 |
| Title |
KO_12.4_U |
| Sample type |
RNA |
| |
|
| Source name |
Mesencymal stem cell line
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Mesencymal stem cell line clone 12.4
genotype: Dicer-/-
differentiation status: undifferentiated
|
| Treatment protocol |
To determine gene expression alterations induced Dicer loss, the cells were infected with Adeno-Cre-GFP, sorted for GFP to enrich for infected cells, and plated at low density for monoclonal isolation. Homozygous deletion within clones was confirmed by genotyping PCR. For isolation of total RNA, cells were passaged under normal culture conditions.
|
| Growth protocol |
Primary mesenchymal stem cells were isolated from the hindlimb bone marrow of a one-year old Dicerflox/flox mouse, transduced with retrovirus encoding SV40 Large T Antigen for immortalization, and then plated at low density for monoclonal isolation. Cells were cultured in Alpha-MEM supplemented with pen/strep and 10% fetal bovine serum. The MIT Institutional Animal Care and Use Committee approved all animal studies and procedures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Qiazol (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Affymetrix GeneChip® WT Sense Target Labeling
|
| |
|
| Hybridization protocol |
Performed according to manufacturer's instructions (Affymetrix) for Affymetrix Mouse Exon 1.0 ST arrays.
|
| Scan protocol |
Affymetrix Gene ChIP Scanner 3000 7G; GeneChip® Command Console.
|
| Data processing |
Data processing was done using Partek Genomics Suite 6.5 software. RMA was used to summarize and normalize the probe data into probe set expression values. The list of utilized probe sets was defined by custom .ps and .mps files. The RMA procedure used included the options adjust for GC content, probe sequence, RMA background correction, Quantile Normalization and median polish probe set summarization. Expression values for probesets were summarized to a single value per gene based on the custom file MoEx-1_0-st-v1._core+miRNA+RfSq+UCSC_.SH.2916.mps. The outlier excluded mean method was used. This procedure excludes probe set values that are more than 3 standard deviations above the mean or more and 2 standard deviations below the mean.
probe group file: MoEx-1_0-st-v1.r2.pgf
probe set file: MoEx-1_0-st-v1._core+miRNA+RfSq+UCSC_.SH.2916.ps
meta-probeset file: MoEx-1_0-st-v1._core+miRNA+RfSq+UCSC_.SH.2916.mps
|
| |
|
| Submission date |
Feb 07, 2013 |
| Last update date |
Apr 10, 2013 |
| Contact name |
A Bhutkar |
| Organization name |
MIT
|
| Street address |
77 Massachusetts Avenue
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL6096 |
| Series (2) |
| GSE44161 |
Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts [Exon array] |
| GSE44163 |
Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts |
|
