|
| Status |
Public on Aug 01, 2008 |
| Title |
Pc_ctrl_embryo_biorep1_techrep2 |
| Sample type |
genomic |
| |
|
| Source name |
whole-embryo chromatin
|
| Organism |
Drosophila melanogaster |
| Characteristics |
0-16h AEL embryo
|
| Treatment protocol |
Chromatin from embryos aged between 0 to 16 h after egg laying was purified as described previously (Birch-Machin et al 2005; Adryan et al. 2007). For the preparation of chromatin from T3 imaginal discs (haltere and third leg) late 3rd instar larvae were dissected in ice-cold Schneider's Medium. Dissected discs were washed with PBS, fixed in PBS/1.5% formaldehyde for 20 min and washed with PBS. Batches of material were snap-frozen in liquid N2 and stored at -80 ºC.
|
| Growth protocol |
Embryo collections were performed at 25 ºC and L3 larvae were raised at 25 ºC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from a minimum of 100 discs or 200mg embryos. Chromatin preparation and ChIP were performed as described previously (Birch-Machin et al. 2005; Adryan et al. 2007). For Pc target analysis the specific reaction used chromatin from the Pc-GFP fly line immunopurified using anti-GFP, and the control reaction used wild type chromatin immunopurified using anti-GFP. For Pho analysis wild type chromatin was used with anti-Pho for the specific reaction and pre-immune antiserum for the control.
|
| Label |
TdT labeled
|
| Label protocol |
10-20 ng of ChIP and control DNA samples were amplified using a random-primed PCR method according to Affymetrix recommendations (Affymetrix Chromatin Immunoprecipitation Assay Protocol; http://www.affymetrix.com/support/technical/manuals.affx). Purified DNAs were then fragmented, TdT labeled, and hybridized to the Affymetrix Drosophila genome Tiling Array 1.0 (reverse part no. 520,054) as described previously (Manak et al. 2006).
|
| |
|
| Hybridization protocol |
Amplified ChIP DNA was fragmented to 50-100bp by treatment with DNAse I for 10min at 37 ºC and 10min at 99 ºC (0.02 U/ug, Epicentre; size distribution of fragmented DNA was verified on a 2% agarose gel). The fragmented DNA was then end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1 ug of fragmented DNA in 1x TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37 ºC. The labeled DNA material was subsequently hybridized to the microarrays for 18 hours at 45 ºC in a 3M TMAC/1X MES-based solution.
|
| Scan protocol |
Chips were scanned on an Affymetrix GeneChip Scanner according to the manufacturer’s recommendations.
|
| Description |
group1: ctrl ChIP using whole-embryo chromatin, anti-GFP in wildtype, chromatin prep 1, hybridisation 2
|
| Data processing |
Sample and control were analysed together in groups (1-4) using the TiMAT package. Affymetrix CEL files were converted into chromosomal enrichment profiles using the TiMAT2 package (http://bdtnp.lbl.gov/TiMAT/TiMAT2/). Probe mapping information (“bpmap”) to D. melanogaster genome release 4 was obtained from David Nix. Each CEL file was visualised for manual inspection and artefacts were masked using CelMasker. Normalisation was subsequently performed with CelProcessor using default parameters. Enrichment profiles were generated using ScanChip, outputting windowed enrichment signals and Wilcoxon Rank Sum scores.
|
| |
|
| Submission date |
Mar 31, 2008 |
| Last update date |
Jun 23, 2008 |
| Contact name |
Steve Russell |
| E-mail(s) |
s.russell@gen.cam.ac.uk
|
| Phone |
+44 (0) 1223 766929
|
| Fax |
+44 (0) 1223 333992
|
| Organization name |
University of Cambridge
|
| Department |
Department of Genetics
|
| Street address |
Downing Street
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 3EH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL5919 |
| Series (1) |
| GSE11006 |
Drosophila Polycomb and Pleiohomeotic genomic binding in 0-16h embryos and L3 larval discs (ChIP-on-chip) |
|
