|
| Status |
Public on Jul 07, 2010 |
| Title |
NA_MES4FLAG_EEMB_2 extraction2_array1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
NA_MES4FLAG_EEMB_2 extraction2_array1 channel_1
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: MES4FLAG tagged by Bombard)
developmental stage: Early Embryo
genotype: mes-4(bn73); bnIs15[mes-3 promoter::MES-4::GFP::3xFLAG::mes-4 3' region
sex: mixed Male and Hermaphrodite population
transgene: mes-3 promoter::MES-4 coding sequence::GFP::3xFLAG::mes-4 3' sequence
|
| Growth protocol |
Worm_embryo_growth_and_harvest_vSS1. 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 100mM Tris-HCl pH 7.5 and once with FA buffer (50 mM HEPES/KOH pH 7.5, 1mM 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/.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_embryo_extraction_vSS1. 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 4 times on ice at the following settings: 30 sec; 20% power output; 0.7 duty cycle. Cell debris was removed by centrifuging at 13,000rpm for 15 minutes at 4ºC and taking the supernatant. Supernatants were filtered through Millipore Ultrafree-MC 0.45 ?m filter units (cat. UFC30HV0S) at 13,000rpm, 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_vSS1. Protein A or G-coupled Dynal magnetic beads from 20 ul of slurry were washed 3 times in 1ml of 5mg/ml IgG-Free BSA in PBS (wash buffer). Beads were resuspended in 100ul of the wash buffer and rotated with 5ug of the appropriate antibody overnight at 4 oC. Beads were further washed 2 times in the wash buffer and resuspended in 30ul of the wash buffer. Before beginning IP, 5% of starting material of protein (3-6mg) was saved as "input" at -20 oC. The antibody-conjugated Dynal beads were mixed with 3-6mg of protein and nutated for 2hrs at 4 oC. After washing the beads, captured proteins were eluted by incubating beads in 300 ul of TE with 1% SDS and 250mM NaCl for 2 times 15 minutes at 65 oC. Samples were treated with proteinase K and reverse-crosslinked at 65 oC overnight. DNA was purified using Zymo DNA purification column and eluted in in 43ul dH2O. RNA was digest for 1 hour at 37 oC with RNase One (Promega). For a detailed protocol see http://www.modencode.org/.
Worm_LM-PCR_Amplification_for_ChIP-chip_vSS1. 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/.
|
| 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.
|
| |
|
| Channel 2 |
| Source name |
NA_MES4FLAG_EEMB_2 extraction2_array1 channel_2
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: MES4FLAG tagged by Bombard)
developmental stage: Early Embryo
genotype: mes-4(bn73); bnIs15[mes-3 promoter::MES-4::GFP::3xFLAG::mes-4 3' region
sex: mixed Male and Hermaphrodite population
transgene: mes-3 promoter::MES-4 coding sequence::GFP::3xFLAG::mes-4 3' sequence
|
| Growth protocol |
Worm_embryo_growth_and_harvest_vSS1. 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 100mM Tris-HCl pH 7.5 and once with FA buffer (50 mM HEPES/KOH pH 7.5, 1mM 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/.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_embryo_extraction_vSS1. 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 4 times on ice at the following settings: 30 sec; 20% power output; 0.7 duty cycle. Cell debris was removed by centrifuging at 13,000rpm for 15 minutes at 4ºC and taking the supernatant. Supernatants were filtered through Millipore Ultrafree-MC 0.45 ?m filter units (cat. UFC30HV0S) at 13,000rpm, 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_vSS1. Protein A or G-coupled Dynal magnetic beads from 20 ul of slurry were washed 3 times in 1ml of 5mg/ml IgG-Free BSA in PBS (wash buffer). Beads were resuspended in 100ul of the wash buffer and rotated with 5ug of the appropriate antibody overnight at 4 oC. Beads were further washed 2 times in the wash buffer and resuspended in 30ul of the wash buffer. Before beginning IP, 5% of starting material of protein (3-6mg) was saved as "input" at -20 oC. The antibody-conjugated Dynal beads were mixed with 3-6mg of protein and nutated for 2hrs at 4 oC. After washing the beads, captured proteins were eluted by incubating beads in 300 ul of TE with 1% SDS and 250mM NaCl for 2 times 15 minutes at 65 oC. Samples were treated with proteinase K and reverse-crosslinked at 65 oC overnight. DNA was purified using Zymo DNA purification column and eluted in in 43ul dH2O. RNA was digest for 1 hour at 37 oC with RNase One (Promega). For a detailed protocol see http://www.modencode.org/.
Worm_LM-PCR_Amplification_for_ChIP-chip_vSS1. 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/.
|
| 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.
|
| |
|
| |
| 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: No Antibody Control;target name: Not Applicable;host: None-Control;antigen: NONE;clonal: None-Control;purified: None-Control;company: None-Control;catalog: Not Applicable;reference: Not Applicable;short description: Specific Antibodies were omitted from this immunoprecipitation reaction (as a negative control).;
|
| 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
|
| |
|
| Submission date |
Jul 05, 2010 |
| Last update date |
Jul 06, 2010 |
| 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 |
GPL7744 |
| Series (1) |
|
