|
| Status |
Public on Jan 27, 2015 |
| Title |
RR(cy3) vs R(cy5) biological replicate 2 dyeswap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
FACS-sorted G1 populations
|
| Organism |
Homo sapiens |
| Characteristics |
cell stage: R
|
| Growth protocol |
Fucci-introduced HCT116 cells were first separated into red-fluorescing G1 (R) and green-fluorescing S/G2/M (G) fractions. The R fraction was cultured for a further 12 h, from which a red-fluorescing G1 population (the red-fluorescingretained‘RR’population) was collected by cell sorting. We chose 12 h for the additional culture duration of R cells, because dividing S/G2/M cells will re-enter a red-fluorescingG1 state and merge with the long-termG1-arrested cells upon further incubation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using miRNeasy kit (Qiagen) following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Differentially labeled 825 ng of each cRNA samples were hybridized on microarray slides. Samples were then hybridized on Agilent 4 × 44K v2 whole human genome arrays (Agilent Design # 039494) at 65 °C for 17 h with rotation in the dark. Hybridization was performed using the Gene Expression Hybridization Kit (Agilent Technologies Inc., Santa Clara, CA, USA) following the manufacturer's instructions.
|
| |
|
| Channel 2 |
| Source name |
FACS-sorted long-term G1 populations
|
| Organism |
Homo sapiens |
| Characteristics |
cell stage: RR
|
| Growth protocol |
Fucci-introduced HCT116 cells were first separated into red-fluorescing G1 (R) and green-fluorescing S/G2/M (G) fractions. The R fraction was cultured for a further 12 h, from which a red-fluorescing G1 population (the red-fluorescingretained‘RR’population) was collected by cell sorting. We chose 12 h for the additional culture duration of R cells, because dividing S/G2/M cells will re-enter a red-fluorescingG1 state and merge with the long-termG1-arrested cells upon further incubation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using miRNeasy kit (Qiagen) following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Differentially labeled 825 ng of each cRNA samples were hybridized on microarray slides. Samples were then hybridized on Agilent 4 × 44K v2 whole human genome arrays (Agilent Design # 039494) at 65 °C for 17 h with rotation in the dark. Hybridization was performed using the Gene Expression Hybridization Kit (Agilent Technologies Inc., Santa Clara, CA, USA) following the manufacturer's instructions.
|
| |
|
| |
| Hybridization protocol |
After washing in GE washing buffer, the slide was scanned with Agilent Microarray Scanner G2505C.
|
| Scan protocol |
Images were quantified using Agilent Feature Extraction Software (version.10.5.1.1).
|
| Description |
RR(cy3) vs R(cy5) biological replicate 2 dyeswap
|
| Data processing |
The scanned images were analyzed with Agilent Feature Extraction Software (v 10.5.1.1) using default parameters.
|
| |
|
| Submission date |
Sep 03, 2014 |
| Last update date |
Jan 27, 2015 |
| Contact name |
Daisuke Okuzaki |
| E-mail(s) |
dokuzaki@biken.osaka-u.ac.jp
|
| Phone |
+81-6-6879-4935
|
| Organization name |
Osaka univ.
|
| Department |
Immunology Frontier Research Center
|
| Lab |
Human Immunology （Single Cell Genomics)
|
| Street address |
Yamadaoka 3-1
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL10332 |
| Series (1) |
| GSE61088 |
The G1 arrest and maintenance mechanism between the long-term G1-arrested and the G1-arrested cells. |
|
