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Sample GSM644511 Query DataSets for GSM644511
Status Public on Dec 22, 2010
Title AB46540_NIGG_N2_MXEMB extraction1_array1 channel_1
Sample type genomic
 
Channel 1
Source name AB46540_NIGG_N2_MXEMB extraction1_array1 channel_1
Organism Caenorhabditis elegans
Characteristics strain: N2
developmental stage: Mixed Embryo
genotype: wild type
Sex: mixed Male and Hermaphrodite population
Growth protocol Worm_embryo_growth_and_harvest_v5. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Live embryos were cross-linked in M9 + 2% formaldehyde for 30 minutes at room temperature followed by quenching with 125mM glycine for 5 minutes. Embryos were then washed twice with M9 Buffer and once by 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_v2. 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 (Calbiochem Cat# 539131). Using a Branson sonifier microtip, samples were sonicated on ice at the following settings: 35% amplitude, 0.9 sec on, 0.1 sec off, 12 pulses, 10 times. Cell debris was removed by centrifuging at 13,000 g for 15 minutes at 4ºC and taking the supernatant. Protein concentration was determined by Bradford Assay and extracts were aliquoted at stored at -80C.
Worm_chromatin_immunoprecipitation_vKI1. Antibody was incubated with protein A/G beads at 4 oC for at least 1 hr and then washed by extraction buffer. For each reaction, 0.5-2 mg of protein extract was taken and sarkosyl was added to 1% final concentration. Before immunoprecipitation, 10% of extract was taken and treated with RNase. The extract was mixed with the antibody-beads complex and incubated overnight at 4C. After washing beads, immune complexes were eluted from beads twice with 150uL elution buffer (1% SDS in TE with 250 mM NaCl) 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. For a detailed protocol see http://www.modencode.org/
Worm_LM-PCR_Amplification_for_ChIP-chip_vKI2. ChIP or input DNA was incubated with T4-DNA polymerase followed by T4-polynucleotide kinase to ensure 5?-phospho and 3?-hydroxyl blunt ends. The DNA fragments were ligated to the double-strand DNA linker with 5?-phosphate and then amplified by using primer corresponding to the linker sequences.
Label Cy3 dye
Label protocol ChIP-chip_label_hyb_nimblegen_v2. DNA was labeled and hybridized to NimbleGen C. elegans tiling array (HD2) 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 with slight modifications. 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 AB46540_NIGG_N2_MXEMB extraction1_array1 channel_2
Organism Caenorhabditis elegans
Characteristics strain: N2
developmental stage: Mixed Embryo
genotype: wild type
Sex: mixed Male and Hermaphrodite population
Growth protocol Worm_embryo_growth_and_harvest_v5. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Live embryos were cross-linked in M9 + 2% formaldehyde for 30 minutes at room temperature followed by quenching with 125mM glycine for 5 minutes. Embryos were then washed twice with M9 Buffer and once by 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_v2. 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 (Calbiochem Cat# 539131). Using a Branson sonifier microtip, samples were sonicated on ice at the following settings: 35% amplitude, 0.9 sec on, 0.1 sec off, 12 pulses, 10 times. Cell debris was removed by centrifuging at 13,000 g for 15 minutes at 4ºC and taking the supernatant. Protein concentration was determined by Bradford Assay and extracts were aliquoted at stored at -80C.
Worm_chromatin_immunoprecipitation_vKI1. Antibody was incubated with protein A/G beads at 4 oC for at least 1 hr and then washed by extraction buffer. For each reaction, 0.5-2 mg of protein extract was taken and sarkosyl was added to 1% final concentration. Before immunoprecipitation, 10% of extract was taken and treated with RNase. The extract was mixed with the antibody-beads complex and incubated overnight at 4C. After washing beads, immune complexes were eluted from beads twice with 150uL elution buffer (1% SDS in TE with 250 mM NaCl) 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. For a detailed protocol see http://www.modencode.org/
Worm_LM-PCR_Amplification_for_ChIP-chip_vKI2. ChIP or input DNA was incubated with T4-DNA polymerase followed by T4-polynucleotide kinase to ensure 5?-phospho and 3?-hydroxyl blunt ends. The DNA fragments were ligated to the double-strand DNA linker with 5?-phosphate and then amplified by using primer corresponding to the linker sequences.
Label Cy5 dye
Label protocol ChIP-chip_label_hyb_nimblegen_v2. DNA was labeled and hybridized to NimbleGen C. elegans tiling array (HD2) 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 with slight modifications. 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_v2. DNA was labeled and hybridized to NimbleGen C. elegans tiling array (HD2) 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 with slight modifications. 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_v2. Array scanning and raw data extraction were performed 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 v2.5 according to the NimbleScan User?s Guide.
Description channel ch1 is input DNA;
channel ch2 is negative control for ChIP; Antibody information listed below:
Data processing ChIP-chip normalization standard MA2C:JL:2 protocol. ChIP-chip_normalization_standard_MA2C_v2. First, all the IP and INPUT log ratio values are read from the pairdata file. Secondly, we build GC bins for INPUT and IP based on the GC counts for every probe sequence, which means that the INPUT or IP values for any probes which have the same GC counts will be put together. After that, for each GC bin, we calculate the mean for IP and INPUT data, and the covariance between this two channels. By default, the robust mean variance method is applied, which generalizes Tukey's theory of bi-weight estimation where the constant C is set to 2. At last, we adjust the log ratio values for each probe by using the mean and covariance values for their corresponding GC bins, then these values are further normalized by their mean and standard derivation. In a single experiment, median within the sliding window defined by 2x bandwidth parameter is assigned as MA2C score at the center of each probe. In case of replicates, when we calculate the MA2Cscore afterwards, we take the median as the score from all the replicates for all the probes within the sliding window defined by 2x bandwidth parameter. Processed data are obtained using following parameters: genome version is WS190
 
Submission date Dec 21, 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
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)
GSE25933 Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2
GSE26200 Lieb AB46540_NIGG_N2_MXEMB
Relations
Named Annotation GSM644511_AB46540_NIGG_N2_MXEMB_1_A_MA2Cscore.wig.gz

Supplementary file Size Download File type/resource
GSM644511_AB46540_NIGG_N2_MXEMB_1_A_294254_NIgG_650_550_532.pair.gz 37.8 Mb (ftp)(http) PAIR
GSM644511_AB46540_NIGG_N2_MXEMB_1_A_294254_NIgG_650_550_635.pair.gz 37.6 Mb (ftp)(http) PAIR
GSM644511_AB46540_NIGG_N2_MXEMB_1_A_MA2Cscore.wig.gz 9.4 Mb (ftp)(http) WIG
GSM644511_AB46540_NIGG_N2_MXEMB_1_A_peaks.gff3.gz 5.0 Kb (ftp)(http) GFF3
Processed data provided as supplementary file
Processed data are available on Series record
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