 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 07, 2010 |
| Title |
SDQ3148_SDC2_N2_MXemb_1 extraction3_array1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
SDQ3148_SDC2_N2_MXemb_1 extraction3_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_v3. 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_v4.1. 2.2 mg extract was taken and sarkosyl was added to 1% final concentration. 0.2 mg 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 4C. Immune complexes were incubated (2hrs at 4C) with 20 uL protein A sepharose (Amersham) and washed 5 minutes 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.
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.
|
| 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 |
SDQ3148_SDC2_N2_MXemb_1 extraction3_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_v3. 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_v4.1. 2.2 mg extract was taken and sarkosyl was added to 1% final concentration. 0.2 mg 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 4C. Immune complexes were incubated (2hrs at 4C) with 20 uL protein A sepharose (Amersham) and washed 5 minutes 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.
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.
|
| 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: SDQ3148 SDC2;target name: SDC-2;host: Rabbit;antigen: 1749-1848 (LIQNEKKLALSHKSHKNDENRRFRLNTVVKWYDAIICHCKEELTQAIVDAFPLNAITKNKETSHVAMENGDDEAMLSDTSDNQMSTTDYQMPKNICRNSE);clonal: Polyclonal;purified: Affinity;company: SDI;catalog: SDI;short description: Antibody was produced by Strategic Diagnostics Inc. SDI to indicated antigen.;
|
| 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 WS180
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 WS180
|
| |
|
| 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
|
| 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 (1) |
|
| Relations |
| Named Annotation |
GSM562688_SDQ3148_SDC2_N2_MXemb_1.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM562688_SDQ3148_SDC2_N2_MXemb_1.wig.gz |
8.5 Mb |
(ftp)(http) |
WIG |
| GSM562688_SDQ3148_SDC2_N2_MXemb_1_14433601_532.pair.gz |
36.7 Mb |
(ftp)(http) |
PAIR |
| GSM562688_SDQ3148_SDC2_N2_MXemb_1_14433601_635.pair.gz |
36.6 Mb |
(ftp)(http) |
PAIR |
| Processed data provided as supplementary file |
|
|
|
|
 |
