|
| Status |
Public on Nov 11, 2007 |
| Title |
Rpb3 antibody untreated 0612_Dm_S2_Rpb3_Untr#2_WG1 2 |
| Sample type |
other |
| |
|
| Channel 1 |
| Source name |
Schneider's cells (S2)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
D. melanogaster S2 cells, male, largely tetraploid
|
| Biomaterial provider |
Drosophila Genomic Resource Center, Indiana University (donor to DGRC: Cherbas Lab)
|
| Treatment protocol |
Untreated
|
| Growth protocol |
Cells were grown in Shields and Sang medium, supplemented with bactopeptone and yeast extract, plus 10% Fetal Bovine Serum, as recommended by the Drosophila Genomics Resource Center
|
| Extracted molecule |
other |
| Extraction protocol |
Cells crosslinked with paraformaldehyde for 10 minutes were re-suspended to 1 ml/108 cells in sonication buffer containing 0.5% SDS and sonicated to produce DNA fragments of 200-500 bp length. Samples were centrifuged to remove cellular debris. After reversal of crosslinks, the DNA recovery was by proteinase-K digestion and phenol-chloroform extraction.
|
| Label |
Cy3
|
| Label protocol |
Immunoprecipitated DNA samples were split into two reactions (~2-5µg/reaction) and each paired with an equimolar amount of WCE DNA labeling reaction performed in parallel. Using the Invitrogen CGH Labeling Kit components, random priming solution was added to a final concentration of 1X in a 75µl reaction. The reaction was mixed by vortexing and placed in a thermal cycler at 95ºC for 5 minutes. The sample was then cooled on ice before the Klenow-containing labeling mixture was added (112/56nM 10X dUTP Nucleotide Mix, 17µM Cy-dUTP, 60U Klenow final concentration). The reaction was mixed and incubated at 37ºC for 5-6 hours. Using the Invitrogen CGH columns, the samples were purified and eluted into 50µl water. The sample was analyzed using a Nanodrop for total DNA yield (6-13µg) and Cy label (4-9 pmol dye).
|
| |
|
| Channel 2 |
| Source name |
Schneider's cells (S2)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
D. melanogaster S2 cells, male, largely tetraploid
|
| Biomaterial provider |
Drosophila Genomic Resource Center, Indiana University (donor to DGRC: Cherbas Lab)
|
| Treatment protocol |
Untreated
|
| Growth protocol |
Cells were grown in Shields and Sang medium, supplemented with bactopeptone and yeast extract, plus 10% Fetal Bovine Serum, as recommended by the Drosophila Genomics Resource Center
|
| Extracted molecule |
other |
| Extraction protocol |
Cells crosslinked with paraformaldehyde for 10 minutes were re-suspended to 1 ml/108 cells in sonication buffer containing 0.5% SDS and sonicated to produce DNA fragments of 200-500 bp length. Samples were centrifuged to remove cellular debris. Material derived from 7.5 x 10^7 cells was immunoprecipitated using an antibody that recognizes the Rpb3 subunit of RNA polymerase II and Protein-A agarose. Washes and DNA recovery were performed as described in Adelman, K. et al. (2005) Mol Cell, 17: pp.103-112.
|
| Label |
Cy5
|
| Label protocol |
Immunoprecipitated DNA samples were split into two reactions (~2-5µg/reaction) and each paired with an equimolar amount of WCE DNA labeling reaction performed in parallel. Using the Invitrogen CGH Labeling Kit components, random priming solution was added to a final concentration of 1X in a 75µl reaction. The reaction was mixed by vortexing and placed in a thermal cycler at 95ºC for 5 minutes. The sample was then cooled on ice before the Klenow-containing labeling mixture was added (112/56nM 10X dUTP Nucleotide Mix, 17µM Cy-dUTP, 60U Klenow final concentration). The reaction was mixed and incubated at 37ºC for 5-6 hours. Using the Invitrogen CGH columns, the samples were purified and eluted into 50µl water. The sample was analyzed using a Nanodrop for total DNA yield (6-13µg) and Cy label (4-9 pmol dye).
|
| |
|
| |
| Hybridization protocol |
Two µg labeled DNA samples from paired immunoprecipitated and input samples were mixed with Human Cot-1 DNA (0.1mg/ml final concentration), Agilent Blocking Agent and Agilent Hybridization Buffer. The mixture was heated to 95ºC for 3 minutes and then incubated at 37ºC for 30 minutes. The samples were spun to collect the samples and 490 µl of sample was loaded onto the array with gasket slide. Hybridization occurred for 40 hours at 65ºC at 10rpm in a rotating hybridization oven. After hybridization, the arrays were disassembled in 6X SSPE and 0.005% N-lauroylsarcosine then transferred to another dish containing a slide rack and 6X SSPE and 0.005% N-lauroylsarcosine. The slides were washed for 5 minutes with stirring and transferred for a few seconds to a container holding 0.06X SSPE. The slides moved again to a fresh solution of 0.06X SSPE and washed with stirring for 5 minutes. The slides were slowly removed from the solution to allow drying. All hybridization and washing steps were performed in an ozone-regulated environment containing less than 1ppb O3.
|
| Scan protocol |
Chips were scanned with an Agilent G2565 Scanner (5micron with XDR). XDR algorithm scans the array twice at two different PMT levels (100% and 10%, H and L respectively in .tif file names) and processed with Agilent Feature Extraction v9.1 (protocol CGH-v4_91).
|
| Description |
Location analysis was conducted using the Drosophila Agilent LA arrays. Starting with immunoprecipitated and WCE DNA, Cy3 and Cy5 labeled DNA was produced using a modified protocol of the Invitrogen CGH labeling kit and purification columns. The labeled DNA samples were mixed as paired (IP to WCE) and hybridized for 40 hours at 65ºC to the probes on the array. Chips were washed using SSPE solutions and scanned using an Agilent scanner. The images were processed with the Agilent Feature Extraction Software to generate .txt files.
|
| Data processing |
Normalized data was obtained by loading the .txt files obtained from Feature Extraction into the Agilent ChIP Analytics software. This software uses several normalization procedures for the default work flow. These include “Blanks subtraction normalization”, “Inter-array median normalization” and “Intra-array (dye-bias) median normalization”. This program also contains an error-model which is used to identify the most likely binding events. This error model uses both a probe-specific intensity based p-value as well as neighbor-specific intensity based p-values to determine a binding event.
|
| |
|
| Submission date |
Dec 26, 2006 |
| Last update date |
Oct 23, 2007 |
| Contact name |
NIEHS Microarray Core |
| E-mail(s) |
[email protected], [email protected]
|
| Organization name |
NIEHS
|
| Department |
DIR
|
| Lab |
Microarray Core
|
| Street address |
111 T.W. Alexander Drive
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL4150 |
| Series (1) |
| GSE6714 |
RNA polymerase is poised for activation across the genome |
|
