|
| Status |
Public on Aug 31, 2013 |
| Title |
SDC-3_smo1RNAi_EMB_rep1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ChIP input DNA from smo-1(RNAi) XX embryo
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: smo-1(RNAi) XX embryo
|
| Treatment protocol |
Briefly, gravid hermaphrodites were bleached and embryos crosslinked with 2% formaldehyde in M9 at room temperature for 30 minutes. Crosslinking was quenched with 100mM Tris-HCl (pH 7.5). Embryos were resuspended and frozen in Homogenization buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF). Samples were sheared with a sonicator.
|
| Growth protocol |
Worms grown on NG agar with HT115 bacteria expressing smo-1 RNAi
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Embryo extracts were prepared as described previously (Jans et al. 2009) except FLAG-tagged SDC-2 (TY2095) ChIP extracts were pre-cleared with protein G sepharose. Briefly, embryos collected by bleaching were crosslinked in 2% formaldehyde in M9 for 30 min at 20oC and quenched with 100 mM Tris-HCl (pH 7.5). Samples were then washed twice with M9, once with Homogenization Buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF) and frozen in liquid nitrogen. 10mM N-Ethylmaleimide (NEM) was added to the Homogenization Buffer used to make extracts for sequential ChIP. Samples were sonicated to a range of 200 bp – 1 kb using a Heat System XL2020 Sonicator with a Misonix 419 tip for 10x30 sec at 10%, then centrifuged at 5,000 x g for 20 minutes at 4°C. The supernatant was again sonicated for 10x30 sec at 10% and spun at 20,000 x g for 20 minutes at 4°C. The resulting supernatant was pre-cleared with protein A sepharose for 30 min and frozen in liquid nitrogen. Immunoprecipitations were performed with the appropriate antibody (as displayed in the beginning of each sample name) and were washed and processed as described in Pferdehirt et al.
|
| Label |
cy3
|
| Label protocol |
3 µg of amplified ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 or Cy5 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| Channel 2 |
| Source name |
SDC-3 ChIP DNA from smo-1(RNAi) XX embryo
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: smo-1(RNAi) XX embryo
|
| Treatment protocol |
Briefly, gravid hermaphrodites were bleached and embryos crosslinked with 2% formaldehyde in M9 at room temperature for 30 minutes. Crosslinking was quenched with 100mM Tris-HCl (pH 7.5). Embryos were resuspended and frozen in Homogenization buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF). Samples were sheared with a sonicator.
|
| Growth protocol |
Worms grown on NG agar with HT115 bacteria expressing smo-1 RNAi
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Embryo extracts were prepared as described previously (Jans et al. 2009) except FLAG-tagged SDC-2 (TY2095) ChIP extracts were pre-cleared with protein G sepharose. Briefly, embryos collected by bleaching were crosslinked in 2% formaldehyde in M9 for 30 min at 20oC and quenched with 100 mM Tris-HCl (pH 7.5). Samples were then washed twice with M9, once with Homogenization Buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF) and frozen in liquid nitrogen. 10mM N-Ethylmaleimide (NEM) was added to the Homogenization Buffer used to make extracts for sequential ChIP. Samples were sonicated to a range of 200 bp – 1 kb using a Heat System XL2020 Sonicator with a Misonix 419 tip for 10x30 sec at 10%, then centrifuged at 5,000 x g for 20 minutes at 4°C. The supernatant was again sonicated for 10x30 sec at 10% and spun at 20,000 x g for 20 minutes at 4°C. The resulting supernatant was pre-cleared with protein A sepharose for 30 min and frozen in liquid nitrogen. Immunoprecipitations were performed with the appropriate antibody (as displayed in the beginning of each sample name) and were washed and processed as described in Pferdehirt et al.
|
| Label |
cy5
|
| Label protocol |
3 µg of amplified ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 or Cy5 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| |
| Hybridization protocol |
Hybridization protocol provided and performed by the FHCRC Genomics Facility using standard NimbleGen protocol
|
| Scan protocol |
Sample scan protocol provided and performed by the FHCRC Genomics Facility using standard NimbleGen protocol
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction.
|
| |
|
| Submission date |
Jun 28, 2013 |
| Last update date |
Aug 31, 2013 |
| Contact name |
Barbara Meyer |
| Organization name |
University of California Berkeley
|
| Department |
Molecular and Cellular Biology
|
| Street address |
16 Barker Hall
|
| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
| |
|
| Platform ID |
GPL8134 |
| Series (1) |
| GSE48413 |
Examination of DPY-30, DPY-27, SDC-3, DPY-26, DPY-28, SDC-2, and SUMOylated DPY-27 binding in wild type embryos and smo-1 RNAi treated embryos |
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