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Sample GSM656312 Query DataSets for GSM656312
Status Public on Jan 17, 2011
Title AB3594_H3K79ME2_346021_N2_L3 extraction1_array1 channel_1
Sample type genomic
 
Channel 1
Source name AB3594_H3K79ME2_346021_N2_L3 extraction1_array1 channel_1
Organism Caenorhabditis elegans
Characteristics strain: N2
developmental stage: L3 Larva
genotype: wild type
Sex: mixed Male and Hermaphrodite population
Growth protocol Worm_L3_growth_and_harvest_vPK1. About 2-7 million of worms are bleached and then hatched in M9 for 24-42 hrs. About 100 embryos are seeded onto the plate to test for contamination and hatching efficiency. Remaining hatched L1 larvae are inoculated in a proper volume of liquid culture. Next day when larvae reach the L3 stage they are cleaned by M9 washes and sucrose gradient and collected by freezing in liquid nitrogen. Just before collection DIC pictures are taken and about 50ul of worms are stained for DAPI to assess the stage.
Extracted molecule genomic DNA
Extraction protocol Worm_L3_extraction_vPK1. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Later, washed pellets are resuspended in FA buffer and subjected to sonication in Bioruptor (14 pulses of 30 seconds with 1 minute rests in between). Extracts are then spun down and soluble fraction is stored for quality tests and future ChIP.
Worm_chromatin_immunoprecipitation_vMBJ6. 2mg 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 (3 ug) was added to each IP sample and incubated overnight at 4C. Immune complexes were incubated (2hrs at 4C) with 50 ul of IgG dynabeads (Dyna), and washed with 1 mL of each of the following solutions: ChIP Buffer, ChIP Buffer+1M NaCl, ChIP Buffer+500mM NaCl, TEL Buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA), and 2X TE (10mM Tris-HCl pH 8.0, 1mM EDTA). Samples were eluted twice with 150uL elution buffer for 15minutes at 65C. Samples were treated with proteinase K at 55C for 1-2hrs then transfer to 65C overnight to reverse crosslinks. DNA was cleaned up Qiagen PCR purification kit and concentrated using by Speed vac. For a detailed protocol see http://www.modencode.org/.
Worm_LM-PCR_Amplification_for_ChIP-chip_v1. ChIP DNA was amplified with a modified ligation mediated PCR (LM-PCR) protocol derived from Ren, R et al (2000) Science 290, 2306-9. Amplification was carried out using 25-30 cycles.
Label Cy3 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.
 
Channel 2
Source name AB3594_H3K79ME2_346021_N2_L3 extraction1_array1 channel_2
Organism Caenorhabditis elegans
Characteristics strain: N2
developmental stage: L3 Larva
genotype: wild type
Sex: mixed Male and Hermaphrodite population
Growth protocol Worm_L3_growth_and_harvest_vPK1. About 2-7 million of worms are bleached and then hatched in M9 for 24-42 hrs. About 100 embryos are seeded onto the plate to test for contamination and hatching efficiency. Remaining hatched L1 larvae are inoculated in a proper volume of liquid culture. Next day when larvae reach the L3 stage they are cleaned by M9 washes and sucrose gradient and collected by freezing in liquid nitrogen. Just before collection DIC pictures are taken and about 50ul of worms are stained for DAPI to assess the stage.
Extracted molecule genomic DNA
Extraction protocol Worm_L3_extraction_vPK1. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Later, washed pellets are resuspended in FA buffer and subjected to sonication in Bioruptor (14 pulses of 30 seconds with 1 minute rests in between). Extracts are then spun down and soluble fraction is stored for quality tests and future ChIP.
Worm_chromatin_immunoprecipitation_vMBJ6. 2mg 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 (3 ug) was added to each IP sample and incubated overnight at 4C. Immune complexes were incubated (2hrs at 4C) with 50 ul of IgG dynabeads (Dyna), and washed with 1 mL of each of the following solutions: ChIP Buffer, ChIP Buffer+1M NaCl, ChIP Buffer+500mM NaCl, TEL Buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA), and 2X TE (10mM Tris-HCl pH 8.0, 1mM EDTA). Samples were eluted twice with 150uL elution buffer for 15minutes at 65C. Samples were treated with proteinase K at 55C for 1-2hrs then transfer to 65C overnight to reverse crosslinks. DNA was cleaned up Qiagen PCR purification kit and concentrated using by Speed vac. For a detailed protocol see http://www.modencode.org/.
Worm_LM-PCR_Amplification_for_ChIP-chip_v1. ChIP DNA was amplified with a modified ligation mediated PCR (LM-PCR) protocol derived from Ren, R et al (2000) Science 290, 2306-9. Amplification was carried out using 25-30 cycles.
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.
 
 
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.
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.
Description channel ch1 is input DNA;
channel ch2 is ChIP DNA; Antibody information listed below: official name: AB3594 H3K79me2:346021;target name: H3K79me2;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 50 to the C-terminus of Human Histone H3 di methylated at K79.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab3594;reference: http://www.abcam.com/Histone-H3-di-methyl-K79-antibody-ChIP-Grade-ab3594.html;short description: Rabbit polyclonal antibody raised against Histone H3 peptide di-methylated at K79.;
Data processing ChIP-chip normalization standard zscore:JL:2 protocol. ChIP-chip_normalization_standard_zscore_v2 First, the ratio of intensity from sample/reference channel was transformed to log2 space. Second, z-scores were obtained for each probe by standardizing the data by subtracting the mean and dividing by the standard deviation of all probes. Processed data are obtained using following parameters: genome version is WS190
 
Submission date Jan 17, 2011
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
Lab modENCODE DCC
Street address MaRS Centre, South Tower, 101 College Street, Suite 800
City Toronto
State/province Ontario
ZIP/Postal code M5G 0A3
Country Canada
 
Platform ID GPL8647
Series (2)
GSE26182 Lieb AB3594_H3K79ME2346021_N2_L3
GSE26186 Broad chromosomal domains of histone modification patterns in C. elegans
Relations
Named Annotation GSM656312_AB3594_H3K79ME2_346021_N2_L3_1_A_MA2Cscore.wig.gz

Supplementary file Size Download File type/resource
GSM656312_AB3594_H3K79ME2346021_N2_L3_1_A_396945_610_510_K79me2_mE5_p_532.pair.gz 38.1 Mb (ftp)(http) PAIR
GSM656312_AB3594_H3K79ME2346021_N2_L3_1_A_396945_610_510_K79me2_mE5_p_635.pair.gz 38.0 Mb (ftp)(http) PAIR
GSM656312_AB3594_H3K79ME2_346021_N2_L3_1_A_MA2Cscore.wig.gz 9.1 Mb (ftp)(http) WIG
GSM656312_AB3594_H3K79ME2_346021_N2_L3_1_A_peaks.gff.gz 44.4 Kb (ftp)(http) GFF
Processed data provided as supplementary file
Processed data are available on Series record

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