|
| Status |
Public on Aug 01, 2010 |
| Title |
H3K36_10day |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ChIP DNA from 10day female heads
|
| Organism |
Drosophila melanogaster |
| Characteristics |
age: 10day
tissue: head
antibody: H3K36me3
antibody manufacturer: Abcam
antibody catalog number: ab9050
strain: Canton-S
|
| Growth protocol |
Mixed sex Canton-S flies were grown in cages for 10 days or 40 days on standard food (6% yeast, 12% sucrose, 5% cornmeal), in a humidified (50%), incubator with 12 hour on/off light cycle at 25˚C, changing food every 2-3 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Flies were kept at -80°C, then sexed, and heads removed by vortexing. 200 mg of heads were used to prepare chromatin (10),with sonication to an average size of 500 bp. 250 µl chromatin extract was immunoprecipitaed with one of the following antibodies: 4H8 (Abcam ab5408, 2 µl/IP), H3K4me3 (Abcam ab8580, 4 µl/IP), H3K9me3 (Millipore 07-442, 4 µl/IP), H3K36me3 (Abcam ab9050, 4 µl/IP), or HP1 (Developmental Studies Hybridoma Bank C1A9, 2 µl/IP). After elution, DNA was purified with the MinElute PCR cleanup kit (Qiagen) and amplified with the WGA2 kit (Sigma).
|
| Label |
biotin
|
| Label protocol |
7.5 ug dsDNA were fragemented for 60 min in standard PCR machine at 37 ºC with 15 U UDG and 225 U APE enzymes, followed by a 2 min incubation at 93 ºC. Successful fragmentation was analyzed on a RNA 6000 Bioanalyzer chip using the total RNA mode. The fragmented samples were labeled with for 60 min at 37 ºC with 1 ul TdT, followed by heat inactivation of the enzyme at 70 ºC for 10 min. Enzymes from fragmentation and labeling were from Affymetrix WT double stranded DNA terminal labeling Kit, (cat # 900812).
|
| |
|
| Channel 2 |
| Source name |
Input DNA from 10day female heads
|
| Organism |
Drosophila melanogaster |
| Characteristics |
age: 10day
tissue: head
antibody: none
strain: Canton-S
|
| Growth protocol |
Mixed sex Canton-S flies were grown in cages for 10 days or 40 days on standard food (6% yeast, 12% sucrose, 5% cornmeal), in a humidified (50%), incubator with 12 hour on/off light cycle at 25˚C, changing food every 2-3 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Flies were kept at -80°C, then sexed, and heads removed by vortexing. 200 mg of heads were used to prepare chromatin (10),with sonication to an average size of 500 bp. 250 µl chromatin extract was immunoprecipitaed with one of the following antibodies: 4H8 (Abcam ab5408, 2 µl/IP), H3K4me3 (Abcam ab8580, 4 µl/IP), H3K9me3 (Millipore 07-442, 4 µl/IP), H3K36me3 (Abcam ab9050, 4 µl/IP), or HP1 (Developmental Studies Hybridoma Bank C1A9, 2 µl/IP). After elution, DNA was purified with the MinElute PCR cleanup kit (Qiagen) and amplified with the WGA2 kit (Sigma).
|
| Label |
biotin
|
| Label protocol |
7.5 ug dsDNA were fragemented for 60 min in standard PCR machine at 37 ºC with 15 U UDG and 225 U APE enzymes, followed by a 2 min incubation at 93 ºC. Successful fragmentation was analyzed on a RNA 6000 Bioanalyzer chip using the total RNA mode. The fragmented samples were labeled with for 60 min at 37 ºC with 1 ul TdT, followed by heat inactivation of the enzyme at 70 ºC for 10 min. Enzymes from fragmentation and labeling were from Affymetrix WT double stranded DNA terminal labeling Kit, (cat # 900812).
|
| |
|
| |
| Hybridization protocol |
The entire labeled sample was used in the hybridization cocktail (200 ul final volume) and the entire sample was hybridized overnight to Drosophila 2.0 tiling arrays following Affymetrix standard protocol.
|
| Scan protocol |
Staining and washing was done on an Affymetrix FS450 Fluidics Station using protocol FS450_001 and array staining was visualized using and Affymetrix 3000 7G scanner.
|
| Description |
10day, H3k36 ChIP Biological Rep 1-3
|
| Data processing |
Background probes were used to calculate median background signal which was subtracted from the signal of each genomic probe based on GC content. All non-unique probe sequences were removed from further analysis. The intensities were quantile normalized together, grouped by antibody. The Hodges-Lehman estimate of triplicate IP samples - triplicate input samples in a 500 bp window centered on the probe was calculated as signal.
|
| |
|
| Submission date |
Jun 18, 2010 |
| Last update date |
Jun 24, 2010 |
| Contact name |
Sara Hillenmeyer |
| E-mail(s) |
[email protected]
|
| Phone |
401-863-2251
|
| Organization name |
Brown University
|
| Department |
MCB
|
| Lab |
Helfand
|
| Street address |
60 Olive St
|
| City |
Providence |
| State/province |
RI |
| ZIP/Postal code |
02906 |
| Country |
USA |
| |
|
| Platform ID |
GPL6629 |
| Series (2) |
| GSE22438 |
Chromatin remodeling in the aging genome of Drosophila |
| GSE22440 |
Chromatin marks and matched expression in the aging Drosophila genome |
|
