|
| Status |
Public on Jul 01, 2013 |
| Title |
Meg01-chIP1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
IgG control
|
| Organism |
Homo sapiens |
| Characteristics |
chip antibody: IgG
cell line: Meg01
chip antibody manufacturer: Santa Cruz
chip antibody catalog #: sc-2028
chip antibody lot #: L2208
|
| Treatment protocol |
The cells used for the chip-on-chip were non-treated Meg-01 cells.
|
| Growth protocol |
The parental Meg01 cells and the shRNA stable clones were cultured in RPMI 1640 with 10% fetal bovine serum (Hyclone, Logan, UT) and 2 mM L-glutamine plus 100 U/ml penicillin and 100 µg/ml streptomycin, in a 37 °C humidified atmosphere containing 5% CO2/95% air and maintained at a density between 1x105 and 8x105 cells/mL.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with formaldehyde, swelled in a hypotonic buffer containing protease inhibitors, and nuclei were isolated. Isolated nuclei were sonicated and an average DNA fragment size of 500-1000bp was verified on an agarose gel. The chromatin solution was precleared by the addtition of protein G beads. The precleared chromatin was incubate with protein G beads and 10 ug of GATA1 (C-20) or normal goat IgG. Immune complexes were eluted, cross-links reversed, and samples were extracted with phenol/chloroform then precipitated. Blunt ends were created and linker DNA was ligated to the blunt ends, then the samples were amplified, labeled and hybridized to the microarray.
|
| Label |
Cy3
|
| Label protocol |
Sample fluorescent labeling started with heating 2 µg amplified control or IP DNA with Random primer at 95°C, followed by cooling on ice and mixing with Exo(-) Klenow fragment master mix. During the incubation of 2 hour at 37°C, control amplified DNA was labeled with Cyanine 3-dUTP, and IP amplified DNA was labeled with Cyanine 5-dUTP. The clean-up of labeled samples was performed using Microcon Ultra YM-30 filters (Millipore, Chicago, IL).
|
| |
|
| Channel 2 |
| Source name |
Meg01 GATA1 IP
|
| Organism |
Homo sapiens |
| Characteristics |
chip antibody: GATA1 (C-20)
cell line: Meg01
chip antibody manufacturer: Santa Cruz
chip antibody catalog #: sc-1233X
chip antibody lot #: I3003
|
| Treatment protocol |
The cells used for the chip-on-chip were non-treated Meg-01 cells.
|
| Growth protocol |
The parental Meg01 cells and the shRNA stable clones were cultured in RPMI 1640 with 10% fetal bovine serum (Hyclone, Logan, UT) and 2 mM L-glutamine plus 100 U/ml penicillin and 100 µg/ml streptomycin, in a 37 °C humidified atmosphere containing 5% CO2/95% air and maintained at a density between 1x105 and 8x105 cells/mL.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with formaldehyde, swelled in a hypotonic buffer containing protease inhibitors, and nuclei were isolated. Isolated nuclei were sonicated and an average DNA fragment size of 500-1000bp was verified on an agarose gel. The chromatin solution was precleared by the addtition of protein G beads. The precleared chromatin was incubate with protein G beads and 10 ug of GATA1 (C-20) or normal goat IgG. Immune complexes were eluted, cross-links reversed, and samples were extracted with phenol/chloroform then precipitated. Blunt ends were created and linker DNA was ligated to the blunt ends, then the samples were amplified, labeled and hybridized to the microarray.
|
| Label |
Cy5
|
| Label protocol |
Sample fluorescent labeling started with heating 2 µg amplified control or IP DNA with Random primer at 95°C, followed by cooling on ice and mixing with Exo(-) Klenow fragment master mix. During the incubation of 2 hour at 37°C, control amplified DNA was labeled with Cyanine 3-dUTP, and IP amplified DNA was labeled with Cyanine 5-dUTP. The clean-up of labeled samples was performed using Microcon Ultra YM-30 filters (Millipore, Chicago, IL).
|
| |
|
| |
| Hybridization protocol |
Hybridization mixture contained 5ug each cyanine 5- and 3-labeled DNA sample in 158µl, 50µl of Human Cot-1 DNA, 50ul of Agilent 10X Blocking Agent and 250 ul of Agilent 2x HI-RPM Hybridization Buffer. After incubation at 95° C for 3 minutes, hyb mixtures were placed in a water bath for the incubation at 37° C for 30 minutes. Samples were immediately loaded to an array in an Agilent SureHyb hybridization chamber and co-hybridize on the array for 40 hours at 65° C and 20rpm. Washing was done using Agilent Oligo aCGH/ ChIP-on-chip Wash buffer 1 and 2 according to the Agilent protocol.
|
| Scan protocol |
Slides were scanned using an Agilent dual laser scanner with extended dynamic range (XDR) at 5 micron resolution.
|
| Data processing |
Tiff images were analyzed with Agilent's feature extraction software (v9.5.3.1) using protocol ChIP-v1_95_May07.
|
| |
|
| Submission date |
Dec 19, 2012 |
| Last update date |
Jul 01, 2013 |
| Contact name |
Yubin Ge |
| E-mail(s) |
gey@karmanos.org
|
| Phone |
313 578-4285
|
| Organization name |
Karmanos Cancer Institute
|
| Street address |
110 East Warren Ave.
|
| City |
Detroit |
| State/province |
MI |
| ZIP/Postal code |
48201 |
| Country |
USA |
| |
|
| Platform ID |
GPL4124 |
| Series (1) |
| GSE43018 |
Identification of GATA1 transcriptional regulation in the megakaryoblastic cell line Meg01 |
|
