|
| Status |
Public on Jan 17, 2011 |
| Title |
AB2621_H3K79ME3_361576_N2_L3 extraction2_array1 channel_1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
AB2621_H3K79ME3_361576_N2_L3 extraction2_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_vIL2. Appropriate amount of extract is incubated overnight with a proper amount of antibody (exceptional antibodies due to better results are incubated 2hrs). Afterwards, 40ul of equilibrated magnetic beads (either protein A or G, depending on antibody) are added and incubated for 2 hrs. Later, washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer are performed and DNA is eluted in elution buffer (1% SDS in TE with 250 mM NaCl) ? two times with 57 ?l volume each, at 65°C. Samples are treated with RNAse, proteinase K and then crosslinks are reversed overnight at 65°C. DNA is purified on Qiagen PCR purification columns, tested by q-PCR for ChIP quality, and stored at -20°C for future applications.
Worm_LM-PCR_Amplification_for_ChIP-chip_vIL1. 1/3 of ChIP and 10ng of input are blunted (T4 polymerase), ligated (concentrated T4 ligase) to annealed linkers and amplified by PCR using longer oligonucleotide as a primer. Generally two rounds of amplification are used to get the amount needed for microarray. Amplified DNA is tested by q-PCR and DNA gel is run to ensure small size and lack of degradation.
|
| 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 |
AB2621_H3K79ME3_361576_N2_L3 extraction2_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_vIL2. Appropriate amount of extract is incubated overnight with a proper amount of antibody (exceptional antibodies due to better results are incubated 2hrs). Afterwards, 40ul of equilibrated magnetic beads (either protein A or G, depending on antibody) are added and incubated for 2 hrs. Later, washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer are performed and DNA is eluted in elution buffer (1% SDS in TE with 250 mM NaCl) ? two times with 57 ?l volume each, at 65°C. Samples are treated with RNAse, proteinase K and then crosslinks are reversed overnight at 65°C. DNA is purified on Qiagen PCR purification columns, tested by q-PCR for ChIP quality, and stored at -20°C for future applications.
Worm_LM-PCR_Amplification_for_ChIP-chip_vIL1. 1/3 of ChIP and 10ng of input are blunted (T4 polymerase), ligated (concentrated T4 ligase) to annealed linkers and amplified by PCR using longer oligonucleotide as a primer. Generally two rounds of amplification are used to get the amount needed for microarray. Amplified DNA is tested by q-PCR and DNA gel is run to ensure small size and lack of degradation.
|
| 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 ChIP DNA; Antibody information listed below: official name: AB2621 H3K79me3:361576;target name: H3K79me3;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 50 to the C-terminus of Human Histone H3 tri methylated at K79.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab2621;reference: http://www.abcam.com/Histone-H3-tri-methyl-K79-antibody-ChIP-Grade-ab2621.html;short description: Rabbit polyclonal antibody raised against Histone H3 peptide tri-methylated at K79.;
channel ch2 is input DNA;
|
| 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
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| |
|
| 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) |
| GSE26183 |
Lieb AB2621_H3K79ME3_361576_N2_L3 |
| GSE26186 |
Broad chromosomal domains of histone modification patterns in C. elegans |
|
| Relations |
| Named Annotation |
GSM656315_AB2621_H3K79ME3_361576_N2_L3_2_A_MA2Cscore.wig.gz |
