|
Status |
Public on Apr 20, 2010 |
Title |
Ls174T si MLLT10 |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
Ls174T colorectal cancer cells
|
Organism |
Homo sapiens |
Characteristics |
cell line: Ls174T transfection: si-MLLT10
|
Treatment protocol |
Control or Wnt3A conditioned medium was added to the HEK293T cells and incubate for 9 hours before RNA extraction
|
Growth protocol |
Ls174T or HEK293T cells were seeded on 6-well plates and transfected with control/human b-catenin/MLLT10/DOT1L/BRG1/P300 15nM siRNA for 72 hours
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA extracted using RNeasy mini kit (Qiagen) following manufacturer's instructions
|
Label |
Cy3
|
Label protocol |
2µg of total RNA were primed with 1.2 µl of T7 promoter primer at 65°C for 10 min together with control spike A/B, then reversed transcribed at 40°C for 2hrs in the presence of MMLV RTase (Agilent), and dNTP mix. It was then followed by the cRNA transcription at 40°C for 2hrs in the presence of T7 RNA polymerase, inorganic pyrophosphatase, NTP mix, DTT, PEG and Cy3/Cy5-labeled-CTP.
|
|
|
Channel 2 |
Source name |
Human reference RNA
|
Organism |
Homo sapiens |
Characteristics |
reference: Human reference RNA
|
Treatment protocol |
Control or Wnt3A conditioned medium was added to the HEK293T cells and incubate for 9 hours before RNA extraction
|
Growth protocol |
Ls174T or HEK293T cells were seeded on 6-well plates and transfected with control/human b-catenin/MLLT10/DOT1L/BRG1/P300 15nM siRNA for 72 hours
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA extracted using RNeasy mini kit (Qiagen) following manufacturer's instructions
|
Label |
Cy5
|
Label protocol |
2µg of total RNA were primed with 1.2 µl of T7 promoter primer at 65°C for 10 min together with control spike A/B, then reversed transcribed at 40°C for 2hrs in the presence of MMLV RTase (Agilent), and dNTP mix. It was then followed by the cRNA transcription at 40°C for 2hrs in the presence of T7 RNA polymerase, inorganic pyrophosphatase, NTP mix, DTT, PEG and Cy3/Cy5-labeled-CTP.
|
|
|
|
Hybridization protocol |
The synthezied cRNA was then purified with RNeasy mini kit (Qiagen). 825ng of the corresponding Cy3 and Cy5 cRNA was fragmentated at 60°C for 30 min in the presence of blocking agent and fragmentation buffer (Agilent), then combined with 55ul 2X hybridization buffer (Agilent) and applied to 4X44k human expression microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially
|
Scan protocol |
Scanned on an Agilent G2505 DNA microarray scanner.
|
Description |
Ls174T cells transfected with si-MLLT10
|
Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
|
|
|
Submission date |
Apr 16, 2010 |
Last update date |
Apr 19, 2010 |
Contact name |
Vivian Li |
E-mail(s) |
vivian.li@crick.ac.uk
|
Organization name |
The Francis Crick Institute
|
Lab |
Stem Cell and Cancer Biology
|
Street address |
1 Midland Road
|
City |
London |
ZIP/Postal code |
NW1 1AT |
Country |
United Kingdom |
|
|
Platform ID |
GPL4133 |
Series (1) |
GSE21367 |
The Leukemia-associated Mllt10/Af10- Dot1l, dedicated b-catenin coactivators essential for intestinal homeostasis |
|