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Status |
Public on Jul 07, 2010 |
Title |
AB8898_H3K9ME3339901_N2_EEMB_1 extraction1_array1 |
Sample type |
genomic |
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Channel 1 |
Source name |
AB8898_H3K9ME3339901_N2_EEMB_1 extraction1_array1 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_vSS3. Embryos were prepared by bleaching from gravid N2 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. For a detailed protocol see http://www.modencode.org/.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Worm_embryo_extraction_vSS2. 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 9 times on ice at the following settings: 30 sec; power output 3; 70% duty cycle. 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_vPK1. Appropriate amount of extract is incubated overnight with a proper amount of antibody (exceptional antibodies due to better results are incubated 2hrs). Afterwards, 40ul of equilibrated magnetic beads (either protein A or G, depending on antibody) are added and incubated for 2 hrs. Later, washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer are performed and DNA is eluted in elution buffer (1% SDS in TE with 250 mM NaCl) ? two times with 57 ?l volume each, at 65°C. Samples are treated with RNAse, proteinase K and then crosslinks are reversed overnight at 65°C. DNA is purified on qiagen PCR purification columns, tested by q-PCR for ChIP quality, and stored in -20°C for future applications. 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 |
Cy5 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 |
AB8898_H3K9ME3339901_N2_EEMB_1 extraction1_array1 channel_2
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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_vSS3. Embryos were prepared by bleaching from gravid N2 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. For a detailed protocol see http://www.modencode.org/.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Worm_embryo_extraction_vSS2. 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 9 times on ice at the following settings: 30 sec; power output 3; 70% duty cycle. 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_vPK1. Appropriate amount of extract is incubated overnight with a proper amount of antibody (exceptional antibodies due to better results are incubated 2hrs). Afterwards, 40ul of equilibrated magnetic beads (either protein A or G, depending on antibody) are added and incubated for 2 hrs. Later, washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer are performed and DNA is eluted in elution buffer (1% SDS in TE with 250 mM NaCl) ? two times with 57 ?l volume each, at 65°C. Samples are treated with RNAse, proteinase K and then crosslinks are reversed overnight at 65°C. DNA is purified on qiagen PCR purification columns, tested by q-PCR for ChIP quality, and stored in -20°C for future applications. 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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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 ChIP DNA; Antibody information listed below: official name: AB8898 H3K9ME3:339901;target name: H3K9me3;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 1-100 of human histone H3 tri methylated at K9.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: AB8898;short description: An affinity purified rabbit polyclonal antibody to H3K9me3 (lot# 339901) obtained from Abcam. Used for ChIP.; channel ch2 is input DNA;
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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
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Phone |
416-673-8579
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Organization name |
Ontario Institute for Cancer Research
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Lab |
modENCODE DCC
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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) |
GSE22725 |
Strome AB8898_H3K9ME3339901_N2_EEMB |
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Relations |
Named Annotation |
GSM562725_AB8898_H3K9ME3339901_N2_EEMB_1.wig.gz |
Supplementary file |
Size |
Download |
File type/resource |
GSM562725_AB8898_H3K9ME3339901_N2_EEMB_1.wig.gz |
9.2 Mb |
(ftp)(http) |
WIG |
GSM562725_OID20832_27331802_532.pair.gz |
36.0 Mb |
(ftp)(http) |
PAIR |
GSM562725_OID20832_27331802_635.pair.gz |
35.6 Mb |
(ftp)(http) |
PAIR |
Processed data provided as supplementary file |
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