|
| Status |
Public on Jul 07, 2010 |
| Title |
WA30834809_H3K4ME2_N2_L3 Hyb1 extraction1_array1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Developmental Stage L3 Larva;temperature 20;Antibody WA308-34809 H3K4me2 (target is H3K4me2);Strain N2 extraction1_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 |
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 |
Developmental Stage L3 Larva;temperature 20;Antibody WA308-34809 H3K4me2 (target is H3K4me2);Strain N2 extraction1_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 |
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 ChIP DNA; Antibody information listed below: official name: WA308-34809 H3K4me2;target name: H3K4me2;host: Mouse;antigen: ART(me2-K)QTARKSTGC;clonal: Monoclonal;purified: Protein A/G;company: Other;catalog: WA308-34809;short description: A protein G purified mouse monoclonal antibody to H3K4me2 (cat #308-34809) obtained from Waco. Used for ChIP.;
|
| 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 |
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) |
| GSE22732 |
Ahringer WA30834809_H3K4ME2_N2_L3 |
| GSE26186 |
Broad chromosomal domains of histone modification patterns in C. elegans |
|
| Relations |
| Named Annotation |
GSM562738_WA30834809_H3K4ME2_N2_L3_1.wig.gz |
