|
| Status |
Public on Mar 14, 2013 |
| Title |
Tet2-ctr |
| Sample type |
RNA |
| |
|
| Source name |
control
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: CaCo2
cell type: colon carcinoma
transfection: shRNA against Myosin V B
|
| Treatment protocol |
Inducible MYO5B RNAi CaCo2 cells were treated for 5 days with 1 µg of Doxycycline obtaining efficient knock down of the myosin Vb protein. Knock down efficiency of induced MYO5B RNAi CaCo2 cells were tested with western blot analysis (supporting information 1).RNA was extracted using the RNeasy kit from Quiagen out of 3 different non-induced and induced MYO5B RNAi CaCo2 cells clone showing equally MYO5B knock down efficiency (supporting information 1). Thereby non-induced MYO5B RNAi CaCo2 cell clones served as controls and induced as knock down samples.
|
| Growth protocol |
CaCo2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing high Glucose supplemented with 10 % heat-inactivated fetal bovine serum, 100 U/ml Penicillin, 100 µg/ml Streptomycin and Sodium Pyruvate. Cells were incubated in a humidified atmosphere with 5 % CO2 at 37 °C. To allow differentiation, CaCo2 cells were grown for 10 days on 24 mm Costar © Transwell filters (pore size 0.4 µm, Corning, Boston, Massachusettes). MYO5B RNAi in CaCo2 cells was performed using lentiviral shRNA against human myosin Vb. Small hairpin RNAs against human MYO5B were generated using the Gateway Multicloning System (Invitrogen). Three different shRNA were designed to facilitate MYO5B knock down. The targeted sequences were as follows: shRNA#1: 5’ggacttacctcttggagaa3’, shRNA#2: 5’aggcagatgatgagaggaa 3’ and shRNA#3: 5’gagaacagcaggacagcaa 3’. All 3 shRNA sequences were first cloned into a pENTR- THT III vector expressing the 5’TO promoter and finally cloned into a pHR – DEST Puro vector. For stably expression of shRNA against human myosin Vb, CaCo2 cells were transfected with the pHR-DEST Puro vector containing the shRNA sequences against human MYO5B and afterwards selected with 12 µg Puromycin (Sigma Aldrich).To generate an inducible MYO5B RNAi cell system, wild type CaCo2 cells were transfected with pLenti-CMV-TetR BLAST plasmid from Addgene for stable expression of the TetR system. Afterwards transfected CaCo2 cells were selected with 12 µg Blasticidin (Invitrogen).CaCo2 cells stably expressing TeTR were cotransfected with pHR-DEST Puro vector containing the shRNA sequences against human myosin Vb and afterwards selected with 12 µg Puromycin (Sigma Aldrich). For infections of CaCo2 cells, lentivirus was packaged into 293T cells. 293T cells were transfected using Lipofectamine LTX plus Reagent (Invitrogen) with the plasmids containing the shRNA sequences together with the vesicular stomatitis virus-G (VSV-G) plasmid and the lentiviral gag-pol packaging vector PAX2. After 48 and 72 hours, respectively, the supernatant containing the virus was harvested and directly used for CaCo2 cell infection. After 48 hours post infection, CaCo2 cells were selected with 12 µg Puromycin (Sigma Aldrich) and 12 µg Blasticidin (Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy kit from Quiagen out of 3 different non-induced and induced MYO5B RNAi CaCo2 cells clone showing equally MYO5B knock down efficiency (supporting information 1). Thereby non-induced MYO5B RNAi CaCo2 cell clones served as controls and induced as knock down samples.
|
| Label |
biotin
|
| Label protocol |
Target RNA was labeled by addition of biotin-linked deoxynucleotides with terminal deoxynucleotidyl transferase according to the manufacturer's (Affymetrix) protocols. Washing and staining was performed in an Affymetrix 450S fluidics station.
|
| |
|
| Hybridization protocol |
Target samples were hybridized to Human Gene 1.0 ST v2 microarrays according to the manufacturer's protocols.The microarrays were washed and stained in an Affymetrix fluidic station 450.
|
| Scan protocol |
Microarrays were scanned in an Affymetrix 3000 scanner.
|
| Data processing |
Raw data were preprocessed using the RMA method. For transcript probe set definition the custom CDF package from the brainarray group (hugene10stv1enstcdf version 13, defining probe sets for transcripts from Ensemble core database version 58) was used.
|
| |
|
| Submission date |
Mar 13, 2013 |
| Last update date |
Mar 14, 2013 |
| Contact name |
Daniel Bindreither |
| E-mail(s) |
daniel.bindreither@gmail.com
|
| Organization name |
Medical University Innsbruck
|
| Department |
Division of Molecular Pathophysiology
|
| Lab |
Applied Bioinformatics Group
|
| Street address |
Innrain 82
|
| City |
Innsbruck |
| State/province |
Tirol |
| ZIP/Postal code |
6020 |
| Country |
Austria |
| |
|
| Platform ID |
GPL16786 |
| Series (1) |
| GSE45134 |
Loss-of-function of MYO5B induces epithelial cell scattering in enterocytes |
|
