|
| Status |
Public on Jul 27, 2012 |
| Title |
Tet2KD_C10_0h_2 |
| Sample type |
RNA |
| |
|
| Source name |
C10 Tet2 knockdown cells, uninduced, rep2
|
| Organism |
Mus musculus |
| Characteristics |
induction time (hours): 0
shRNA: Tet2
cell line origin: HAFTL
|
| Treatment protocol |
Induction was carried out in the growth media supplemented with 100 nM bEstradiol, 10 ng/ml IL-3 and 10 ng/ml M-CSF.
|
| Growth protocol |
C10 cells were maintained in RPMI media supplemented with 10% FBS, 20mM L-glutamine, 20μM 2-mercaptoethanol & 1μg/ml puromycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using RNeasy spin columns (Qiagen catalogue number 74104) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA from each sample were amplified by Oligo-dT-T7 reverse transcription and labeled by in vitro transcription with T7 RNA polymerase in the presence of Cy3-CTP using the Quick Amp Labeling kit (Agilent) and purified using RNAeasy columns (Qiagen, Hilden, Germany).
|
| |
|
| Hybridization protocol |
After fragmentation, 1650 ng of labeled cRNA from each sample was hybridized in in situ hybridization oven (Agilent) for 17 h at 65ºC and washed during 1 min at rt in Gene Expression Wash Buffer 1 (Agilent) and1 min at 37 ºC with Gene Expression Wash buffer 2 (Agilent).
|
| Scan protocol |
Scanned on an Agilent G2539A scanner at 5um resolution and 110%PMT. The intensity data of each individual hybridization were extracted and the quality was assessed with the Feature Extraction software 10.7 (Agilent).
|
| Description |
Gene expression in uninduced C10 cells expressing shRNA targeting Tet2 mRNA
No primary cells were used in this study - only derivatives of an established cell line called HAFTL
|
| Data processing |
Raw data was corrected for background noise using the normexp method. Quantile normalization was applied to assure comparability across samples. Data were normalized using the limma package in Bioconductor version 2.8.
|
| |
|
| Submission date |
Jul 26, 2012 |
| Last update date |
Jul 27, 2012 |
| Contact name |
Eric M. Kallin |
| E-mail(s) |
eric.kallin@crg.es
|
| Organization name |
Center for Genomic Regulation
|
| Department |
Differentiation and Cancer
|
| Lab |
Thomas Graf
|
| Street address |
C/ Dr. Aiguader, 88
|
| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10333 |
| Series (1) |
| GSE39666 |
Tet2 facilitates the de-repression of myeloid target genes during C/EBPa induced transdifferentiation of pre-B cells |
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