|
| Status |
Public on Sep 07, 2011 |
| Title |
OregonR^TOW Stage 11/12 MCM2-7 ChIP |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
MCM2-7 ChIP DNA from OregonR^TOW pooled stage 11 and 12 egg chambers
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: egg chambers
strain: OregonR^TOW
chip antibody: MCM2-7
|
| Treatment protocol |
Stage 11 and 12 egg chambers were hand sorted from 2-3 day fattened female flies
|
| Growth protocol |
Drosophila were grown on standard cornmeal, molasses agar media
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Staged egg chambers were fixed with 2% formaldehyde in Grace's medium for 15 min at room temperature followed by quenching with 0.125 M glycine for 5 min (4 samples of 300 egg chambers each). The cells were washed twice with TBS 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 5 uL of MCM2-7 antibody (Steve Bell, MIT) 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. DNAs from four samples were pooled together. 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
|
| |
|
| Channel 2 |
| Source name |
Input DNA from OregonR^TOW Stage 11 and 12 egg chambers
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: egg chambers
strain: OregonR^TOW
chip antibody: none; input DNA
|
| Treatment protocol |
Stage 11 and 12 egg chambers were hand sorted from 2-3 day fattened female flies
|
| Growth protocol |
Drosophila were grown on standard cornmeal, molasses agar media
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Staged egg chambers were fixed with 2% formaldehyde in Grace's medium for 15 min at room temperature followed by quenching with 0.125 M glycine for 5 min (4 samples of 300 egg chambers each). The cells were washed twice with TBS 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 5 uL of MCM2-7 antibody (Steve Bell, MIT) 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. DNAs from four samples were pooled together. 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
|
| |
|
| |
| 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 |
Sep 07, 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 |
GPL11290 |
| Series (2) |
| GSE31889 |
ChIP-chip from Drosophila egg chambers using MCM2-7 antibody |
| GSE31891 |
Analysis of gene amplification at DAFC-34B |
|
