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| Status |
Public on Mar 09, 2011 |
| Title |
SDQ3891_LEM2_SP646_MXEMB extraction1_array1 channel_1 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
SDQ3891_LEM2_SP646_MXEMB extraction1_array1 channel_1
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: SP646
developmental stage: Mixed Embryo
genotype: mnT12(IV;X).
Sex: mixed Male and Hermaphrodite population
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| 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.
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| 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_vKI3. 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. 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 RNase A at room temperature for ? 1 hr then proteinase K at 55C for ? 1 hr. Samples were transferred 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_vKI3. DNA was incubated with End Repair Enzyme mix (Epicentre, Madison) to ensure blunt ends and then with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends. The DNA fragments were ligated with T-over-hanged linker and then amplified by PCR with the longer linker oligonucleotide as a primer.
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| Label |
Cy3 dye
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| 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.
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| Channel 2 |
| Source name |
SDQ3891_LEM2_SP646_MXEMB extraction1_array1 channel_2
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| Organism |
Caenorhabditis elegans |
| Characteristics |
Strain: SP646
Developmental Stage: Mixed Embryo
Genotype: mnT12(IV;X).
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_vKI3. 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. 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 RNase A at room temperature for ? 1 hr then proteinase K at 55C for ? 1 hr. Samples were transferred 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_vKI3. DNA was incubated with End Repair Enzyme mix (Epicentre, Madison) to ensure blunt ends and then with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends. The DNA fragments were ligated with T-over-hanged linker and then amplified by PCR with the longer linker oligonucleotide as a primer.
|
| 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.
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| |
| 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.
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| 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.
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| Description |
channel ch1 is input DNA;
channel ch2 is ChIP DNA; Antibody information listed below: official name: SDQ3891 LEM2;target name: LEM-2;host: Rabbit;antigen: MVDVEKMSDA ELRAELNVRG ANVGPVTGTT RSLYEKKLKK LLSGGAKTPA RPTVAKPAPK PTPKSAPAPK SPKSPPARRS IPRAAATAAN STINSTFNRS ; clonal: Polyclonal;purified: Affinity;company: SDI;catalog: SDQ3891 LEM2;short description: Rabbit antisera against the amino-terminal 100 amino acids of LEM-2 produced at SDI.;
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| 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
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| Submission date |
Mar 07, 2011 |
| Last update date |
Mar 26, 2012 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
help@modencode.org
|
| Phone |
416-673-8579
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| Organization name |
Ontario Institute for Cancer Research
|
| 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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| Supplementary file |
Size |
Download |
File type/resource |
| GSM686623_SDQ3891_LEM2_SP646_MXEMB_1_A_SP646_KI1777_387652s_LEM2_SP646_620_530_532.pair.gz |
223 b |
(ftp)(http) |
PAIR |
| GSM686623_SDQ3891_LEM2_SP646_MXEMB_1_A_SP646_KI1777_387652s_LEM2_SP646_620_530_635.pair.gz |
223 b |
(ftp)(http) |
PAIR |
| GSM686623_SDQ3891_LEM2_SP646_MXEMB_1_A_SP646_KI1777_peaks.gff3.gz |
198 b |
(ftp)(http) |
GFF3 |
| Processed data provided as supplementary file |
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