|
| Status |
Public on Dec 22, 2011 |
| Title |
Drosophila-Cbx-replicate1 (Test vs Mock) [Agilent-014817] |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
[MockIP_WCE] CbxHM/+ wing discs
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype/variation: CbxHm/+
developmental stage: Third Instar larvae
tissue: wing discs
|
| Growth protocol |
Wildtype (CS) flies were crossed to flies of CbxHm/TM6B genotype and reared at 25oC. From resultant progeny CbxHm/+ wing discs were manually dissected in chilled PBS and fixed in 1% formaldehyde solution at room temperature. Chromatin was sheared to obtain average 500 bp fragments and pulled down using polyclonal antibodies generated against N-terminal region of Ubx
1:500 dilution of Ubx antibody was used to carry out pull down in 300 µl volume. Upstate Ez ChIP kit (Catalog # 17-371) was used for binding and washing the immune complex as per manufacturer's instructions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was eluted and processed as per Agilent ChIP-on-chip protocol available at www.agilent.com
|
| Label |
Cy3
|
| Label protocol |
The LMPCR samples (1.5 micrograms each) were labeled in replicates using Agilent Genomic DNA Labeling Kit PLUS and Cy3 or Cy5 dUTP from Agilent. DNA yield and incorporation of label (specific activity) was measured using NanoDrop spectrophotometer. For all hybridizations, 5 micrograms each of Cy3-labeled Mock IP (labelled as WCE in raw data) was mixed with 5 micrograms of Cy5-labeled Test-IP DNA
|
| |
|
| Channel 2 |
| Source name |
[ChIP fraction] CbxHM/+ wing discs
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype/variation: CbxHm/+
developmental stage: Third Instar larvae
tissue: wing discs
chip antibody: anti-Ubx (against N-terminal region of Ubx protein)
chip antibody supplier: generated in-house
|
| Growth protocol |
Wildtype (CS) flies were crossed to flies of CbxHm/TM6B genotype and reared at 25oC. From resultant progeny CbxHm/+ wing discs were manually dissected in chilled PBS and fixed in 1% formaldehyde solution at room temperature. Chromatin was sheared to obtain average 500 bp fragments and pulled down using polyclonal antibodies generated against N-terminal region of Ubx
1:500 dilution of Ubx antibody was used to carry out pull down in 300 µl volume. Upstate Ez ChIP kit (Catalog # 17-371) was used for binding and washing the immune complex as per manufacturer's instructions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was eluted and processed as per Agilent ChIP-on-chip protocol available at www.agilent.com
|
| Label |
Cy5
|
| Label protocol |
The LMPCR samples (1.5 micrograms each) were labeled in replicates using Agilent Genomic DNA Labeling Kit PLUS and Cy3 or Cy5 dUTP from Agilent. DNA yield and incorporation of label (specific activity) was measured using NanoDrop spectrophotometer. For all hybridizations, 5 micrograms each of Cy3-labeled Mock IP (labelled as WCE in raw data) was mixed with 5 micrograms of Cy5-labeled Test-IP DNA
|
| |
|
| |
| Hybridization protocol |
Using Agilent’s In situ Hybridzation kit 5184-3568. As per Manufacturer's instructions.
|
| Scan protocol |
Post-hybridization, the slides were washed as per Agilent protocol and scanned at 5 micron resolution
using Agilent Microarray Scanner.
|
| Description |
Rep1_Agilent-014817
Biological replicate 1 of 3 slide 2, Cbx discs, Ubx ChIP
Agilent Feature Extraction File: US45103024_251481710121_S01.txt
|
| Data processing |
Agilent Feature Extraction Software (v 10.2) was used for background subtraction and LOWESS normalization. The maximun distance(bp) for two probes to be considered as neighbors was taken as 500 bp in chip analytics software. The updated Agilent design file was used to extract the data Build April 2006. (dm3).
|
| |
|
| Submission date |
Apr 21, 2011 |
| Last update date |
Dec 23, 2011 |
| Contact name |
Pavan Agrawal |
| E-mail(s) |
pavan.agrawal123@gmail.com
|
| Phone |
+912025908001
|
| Organization name |
Indian Institute of Science Education and Research (IISER)
|
| Department |
Biology
|
| Lab |
L. S. Shashidhara
|
| Street address |
First floor, Central Tower, Sai Trinity Building, Pashan
|
| City |
Pune |
| State/province |
Maharashtra |
| ZIP/Postal code |
411021 |
| Country |
India |
| |
|
| Platform ID |
GPL4150 |
| Series (1) |
| GSE28778 |
Genome level identification of direct Ubx targets from third-instar imaginal discs |
|
