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| Status |
Public on Jan 24, 2012 |
| Title |
Pol II_met-1_rep2 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
Pol II ChIP DNA for met-1 EEMB
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: met-1(n4337)
developmental stage: Early Embryo
genotype: met-1(n4337)
chip antibody: Pol II
Sex: population predominantly Hermaphrodites perhaps with some Males
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| Growth protocol |
Worm_embryo_growth_and_harvest_vSS3. Embryos were prepared by bleaching from gravid met-1 adults grown in standard S-basal media liquid culture. Live embryos were cross-linked in M9 + 1.85 % formaldehyde for 30 minutes at room temperature. Embryos were then washed twice with M9 Buffer, once with 100 mM Tris-HCl pH 7.5 and once with FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl). Pellets were frozen at -80C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_embryo_extraction_vSS5. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl) + protease inhibitors. Sample was dounced 30 times using tight pestle at 4ºC, then sonicated in ice water bath using a Diagenode Bioruptor at the highest setting for 14 pulses 30 seconds long with 1 minute pauses. Cell debris was removed by centrifuging at 13,000 rpm for 15 minutes at 4ºC and taking the supernatant. Supernatants were filtered through Millipore Ultrafree-MC 0.45 um filter units (cat. UFC30HV0S) at 13,000 rpm, 4°C for 1 minute to remove lipids. Protein concentration was determined, and extracts were aliquoted and stored at -80ºC. Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ul of IgG dynabeads (Dyna), and washed 5 minutes with 1.5 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 uL elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Zymo kit. Worm_LM-PCR_Amplification_for_ChIP-chip_vSS2. ChIP DNA was amplified with a modified ligation mediated PCR (LM-PCR) protocol derived from FlyChip protocol version 1.2 from R. Auburn: http://www.flychip.org.uk/protocols/chip/lm_pcr.php. For more details see www.modencode.org
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| Label |
Cy5
|
| Label protocol |
ChIP-chip_label_hyb_nimblegen_v1. DNA was labeled and hybridized to C. elegans tiling array by Roche NimbleGen according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays Users Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, Amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.
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| Channel 2 |
| Source name |
Input DNA for met-1 EEMB
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: met-1(n4337)
developmental stage: Early Embryo
genotype: met-1(n4337)
chip antibody: none, Input DNA
Sex: population predominantly Hermaphrodites perhaps with some Males
|
| Growth protocol |
Worm_embryo_growth_and_harvest_vSS3. Embryos were prepared by bleaching from gravid met-1 adults grown in standard S-basal media liquid culture. Live embryos were cross-linked in M9 + 1.85 % formaldehyde for 30 minutes at room temperature. Embryos were then washed twice with M9 Buffer, once with 100 mM Tris-HCl pH 7.5 and once with FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl). Pellets were frozen at -80C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_embryo_extraction_vSS5. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl) + protease inhibitors. Sample was dounced 30 times using tight pestle at 4ºC, then sonicated in ice water bath using a Diagenode Bioruptor at the highest setting for 14 pulses 30 seconds long with 1 minute pauses. Cell debris was removed by centrifuging at 13,000 rpm for 15 minutes at 4ºC and taking the supernatant. Supernatants were filtered through Millipore Ultrafree-MC 0.45 um filter units (cat. UFC30HV0S) at 13,000 rpm, 4°C for 1 minute to remove lipids. Protein concentration was determined, and extracts were aliquoted and stored at -80ºC. Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ul of IgG dynabeads (Dyna), and washed 5 minutes with 1.5 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 uL elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Zymo kit. Worm_LM-PCR_Amplification_for_ChIP-chip_vSS2. ChIP DNA was amplified with a modified ligation mediated PCR (LM-PCR) protocol derived from FlyChip protocol version 1.2 from R. Auburn: http://www.flychip.org.uk/protocols/chip/lm_pcr.php. For more details see www.modencode.org
|
| Label |
Cy3
|
| Label protocol |
ChIP-chip_label_hyb_nimblegen_v1. DNA was labeled and hybridized to C. elegans tiling array by Roche NimbleGen according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays Users Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, Amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.
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| |
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| |
| Hybridization protocol |
ChIP-chip_label_hyb_nimblegen_v1. DNA was labeled and hybridized to C. elegans tiling array by Roche NimbleGen according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, Amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.
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| Scan protocol |
ChIP-chip_scanning_nimblegen_v1. Array scanning and raw data extraction were performed at Roche NimbleGen, according to the protocol described in chapter 5 and 6 of the NimbleGen Arrays Users Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, array signal was scanned by using a GenePix 4000B Scanner with associated software and saved as .tif files of the 532nm and 635nm images individually. Raw signal intensities of the images were extracted and saved as .pair files by using NimbleScan software according to the NimbleScan v2.4 User?s Guide.
|
| Description |
ChIP-chip of Pol II in met-1 EEMB; channel ch1 is ChIP DNA; ChIP-chip of CENP-A/HCP-3 in met-1 EEMB; channel ch1 is ChIP DNA; Antibody information: official name: ABAB817 8WG16;target name: RNA polII CTD domain unphosophorylated;host: Mouse;antigen: Native RNA Polymerase II purified from wheat germ;clonal: Monoclonal;purified: Size;company: Covance;catalog: Covance (MMS126R);short description: A mouse monoclonal antibody which recognizes primarily the unphosphorylated RNA Pol II CTD (8WG16) obtained from Covance (MMS126R) was used for ChIP; channel ch2 is Input DNA;
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| Data processing |
ChIP-chip normalization standard nimblegen:JL:1 protocol. The log2-ratio ratio of intensity from sample/reference channel was scaled to center the ratio data around zero by subtracting the bi-weight mean for the log2-ratio values for all features on the array from each log2-ratio value (results in linked .gff files). This was performed using NimbleScan software according to the NimbleScan v2.4 User's Guide and chapter 6 of the NimbleGen Arrays User's Guide ChIP-chip Analysis (version 3.1, 27 May 2008) at Roche NimbleGen. Processed data are obtained using following parameters: genome version is WS180
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| Submission date |
Jan 23, 2012 |
| Last update date |
Jan 24, 2012 |
| Contact name |
Susan Strome |
| E-mail(s) |
sstrome@ucsc.edu
|
| Organization name |
UC Santa Cruz
|
| Department |
MCD Biology
|
| Street address |
1156 High St
|
| City |
Santa Cruz |
| State/province |
CA |
| ZIP/Postal code |
95064 |
| Country |
USA |
| |
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| Platform ID |
GPL8647 |
| Series (1) |
| GSE35079 |
Strome Pol II, H3K36me3 and CENP-A in met-1 EEMB |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM864794_8WG16.met1_O34991_69171502_532.Cy3.pair.gz |
36.2 Mb |
(ftp)(http) |
PAIR |
| GSM864794_8WG16.met1_O34991_69171502_635.Cy5.pair.gz |
35.6 Mb |
(ftp)(http) |
PAIR |
| GSM864794_8WG16.met1_O34991_69171502_ratio.gff.gz |
29.1 Mb |
(ftp)(http) |
GFF |
| Processed data provided as supplementary file |
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