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| Status |
Public on Jul 07, 2010 |
| Title |
UP07442_H3K9ME3_N2_EEMB_3_OID18184 extraction3_array2 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
UP07442_H3K9ME3_N2_EEMB_3_OID18184 extraction3_array2 channel_1
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
developmental stage: Early Embryo
genotype: wild type
sex: mixed Male and Hermaphrodite population
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| Growth protocol |
Worm_embryo_growth_and_harvest_vSS6. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Embryos were then washed 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. For a detailed protocol see http://www.modencode.org/.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_embryo_extraction_vSS4. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM 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 fixed for 10 min with formaldehyde. Nuclei were isolated and then sonicated 14 times in ice water bath followed by 1 minute pause at the highest setting. 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 ?m 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. For a detailed protocol see http://www.modencode.org/.
Worm_chromatin_immunoprecipitation_vSS2. 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) and treated with RNAse. Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ?l 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 ?L elution buffer for 15 minutes at 65ºC. 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 purification kit. For a detailed protocol see http://www.modencode.org/.
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 a detailed protocol see http://www.modencode.org/.
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| Label |
Cy3 dye
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| 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 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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| Channel 2 |
| Source name |
UP07442_H3K9ME3_N2_EEMB_3_OID18184 extraction3_array2 channel_2
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
developmental stage: Early Embryo
genotype: wild type
sex: mixed Male and Hermaphrodite population
|
| Growth protocol |
Worm_embryo_growth_and_harvest_vSS6. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Embryos were then washed 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. For a detailed protocol see http://www.modencode.org/.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_embryo_extraction_vSS4. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM 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 fixed for 10 min with formaldehyde. Nuclei were isolated and then sonicated 14 times in ice water bath followed by 1 minute pause at the highest setting. 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 ?m 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. For a detailed protocol see http://www.modencode.org/.
Worm_chromatin_immunoprecipitation_vSS2. 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) and treated with RNAse. Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ?l 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 ?L elution buffer for 15 minutes at 65ºC. 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 purification kit. For a detailed protocol see http://www.modencode.org/.
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 a detailed protocol see http://www.modencode.org/.
|
| Label |
Cy5 dye
|
| 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 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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| 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 User?s 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.
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| Description |
channel ch1 is input DNA;
channel ch2 is ChIP DNA; Antibody information listed below: official name: C. elegans UP07442 H3K9ME3 rabbit polyclonal antibody;target name: Trimethylated Lys-9 on histone H3;host: Rabbit;antigen: synthetic 2X-branched peptide containing the sequence AR[me3K]ST in which me3K corresponds to trimethyl-lysine at residue 9 of human Histone H3;clonal: Polyclonal;purified: Protein A/G;company: Upstate;catalog: UP07442;short description: A protein A purified rabbit polyclonal antibody to H3K9me3 obtained from Upstate (H3K9me3-07442);used for ChIP.;
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| Data processing |
ChIP-chip normalization standard nimblegen:JL:1 protocol. ChIP-chip_normalization_standard_nimblegen_v1. 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. 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
ChIP-chip normalization standard nimblegen:JL:1 protocol. ChIP-chip_normalization_standard_nimblegen_v1. 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. 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 |
Jul 05, 2010 |
| Last update date |
Feb 02, 2015 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
help@modencode.org
|
| Phone |
416-673-8579
|
| Organization name |
Ontario Institute for Cancer Research
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| Lab |
modENCODE DCC
|
| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
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| Platform ID |
GPL8647 |
| Series (1) |
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| Relations |
| Named Annotation |
GSM562694_UP07442_H3K9ME3_N2_EEMB_3.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM562694_UP07442_H3K9ME3_N2_EEMB_3.wig.gz |
9.2 Mb |
(ftp)(http) |
WIG |
| GSM562694_UP07442_H3K9ME3_N2_EEMB_3_OID18184_14631801_532.pair.gz |
36.7 Mb |
(ftp)(http) |
PAIR |
| GSM562694_UP07442_H3K9ME3_N2_EEMB_3_OID18184_14631801_635.pair.gz |
35.7 Mb |
(ftp)(http) |
PAIR |
| Processed data provided as supplementary file |
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