|
| Status |
Public on Oct 18, 2011 |
| Title |
SuUR 3SG ORC2 ChIP 1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
orc2 ChIP DNA from third instar salivary gland from SuUR
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: third instar salivary gland
strain: SuUR-
antibody: orc2 (Steve Bell, MIT)
|
| Treatment protocol |
Salivary glands were hand dissected from committed wandering third instar larvae directly into EBR medium with protease inhibitors on ice. After no more than 30 minutes samples were fixed and processed as described below.
|
| Growth protocol |
Drosophila were grown on standard cornmeal, molasses agar media.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Salivary glands were fixed with 2% formaldehyde in EBR medium for 15 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with TBS and flash frozen in liquid nitrogen. Approximately 200 salivary glands were thawed, pooled, and placed in 0.5 mL of ChIP lysis buffer (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate). The crosslinked chromatin was sheared to an average size of 300-1000 bp by three 12-second pulses using a Branson 250 sonicator. The lysate was then centrifuged to remove cell debris. 50 uL was saved for the input DNA from each sample. The chromatin supernatant was then incubated overnight with 2 uL of antibody at 4C. After incubation with 30 uL protein G Dynabeads beads (Invitrogen) for 2h at 4C, the immune complexes were collected using the Dynal magnetic strip and washed with the following buffers at room temperature for 5 minutes each: 2X ChIP lysis buffer, 2X high salt (500mM NaCl) ChIP lysis buffer, 1X ChIP lysis buffer, 1X TE. The protein-DNA complexes were eluted from the beads in 100 uL ChIP elution buffer (50 mM Tris/HCl pH 8.0, 10 mM EDTA/1% SDS) at 65C for 15 min followed by another wash with 150 uL TE/0.67% SDS. Eluates were pooled and incubated at 65C for at least 6 hours. Input DNAs were similarly subject to reversal of crosslinks with 200 µL TE/1% SDS. Samples were treated with 100 ug proteinase K at 37C for 2 hours. DNA samples were phenol chloroform extracted twice and ethanol precipitated.
|
| Label |
Cy5
|
| Label protocol |
DNA was labeled using Invitrogen's BioPrime Total for Agilent aCGH labeling kit
|
| |
|
| Channel 2 |
| Source name |
Input DNA from SuUR salivary glands
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: third instar salivary gland
strain: SuUR-
antibody: n/a
|
| Treatment protocol |
Salivary glands were hand dissected from committed wandering third instar larvae directly into EBR medium with protease inhibitors on ice. After no more than 30 minutes samples were fixed and processed as described below.
|
| Growth protocol |
Drosophila were grown on standard cornmeal, molasses agar media.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Salivary glands were fixed with 2% formaldehyde in EBR medium for 15 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with TBS and flash frozen in liquid nitrogen. Approximately 200 salivary glands were thawed, pooled, and placed in 0.5 mL of ChIP lysis buffer (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate). The crosslinked chromatin was sheared to an average size of 300-1000 bp by three 12-second pulses using a Branson 250 sonicator. The lysate was then centrifuged to remove cell debris. 50 uL was saved for the input DNA from each sample. The chromatin supernatant was then incubated overnight with 2 uL of antibody at 4C. After incubation with 30 uL protein G Dynabeads beads (Invitrogen) for 2h at 4C, the immune complexes were collected using the Dynal magnetic strip and washed with the following buffers at room temperature for 5 minutes each: 2X ChIP lysis buffer, 2X high salt (500mM NaCl) ChIP lysis buffer, 1X ChIP lysis buffer, 1X TE. The protein-DNA complexes were eluted from the beads in 100 uL ChIP elution buffer (50 mM Tris/HCl pH 8.0, 10 mM EDTA/1% SDS) at 65C for 15 min followed by another wash with 150 uL TE/0.67% SDS. Eluates were pooled and incubated at 65C for at least 6 hours. Input DNAs were similarly subject to reversal of crosslinks with 200 µL TE/1% SDS. Samples were treated with 100 ug proteinase K at 37C for 2 hours. DNA samples were phenol chloroform extracted twice and ethanol precipitated.
|
| Label |
Cy3
|
| Label protocol |
DNA was labeled using Invitrogen's BioPrime Total for Agilent aCGH labeling kit
|
| |
|
| |
| Hybridization protocol |
Slides were hybridized to custom Agilent tiling arrays and washed as per Agilent recommendations
|
| Scan protocol |
Arrays were scanned on an Agilent scanner per manufacturer's protocol
|
| Data processing |
Raw data was median normalized using the Ringo package in R in order to generate .wig files containing smoothed log ratios.
|
| |
|
| Submission date |
Sep 06, 2011 |
| Last update date |
Oct 18, 2011 |
| Contact name |
Terry L. Orr-Weaver |
| E-mail(s) |
weaver@wi.mit.edu
|
| Phone |
617-258-5251
|
| Organization name |
Whitehead Institute for Biomedical Research
|
| Lab |
Orr-Weaver
|
| Street address |
9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL7787 |
| Series (2) |
| GSE31895 |
ChIP with anti-orc2 antibody to identify regions of orc binding in third instar salivary glands of WT and SuUR mutant Drosophila |
| GSE31900 |
Developmental Control of Gene Copy Number by Repression of Replication Initiation and Fork Progression |
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