|
| Status |
Public on Aug 15, 2016 |
| Title |
LEM2 ChIP in IMB-1 RNAi embryo KI1967 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
SDQ4051_LEM2_RiIMB1_KI1967
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
treatment: IMB-1 RNAi
chip antibody: SDQ4051_LEM2 [Novus Biologicals, catalog# 48540002, lot# G3048-198A02, Animal Q4051]
|
| Treatment protocol |
The N2 strain worms were arrested at the L1 larva stage in the liquid S medium for synchronization. Synchronized L1 worms were fed with a bacteria strain HB101, which do not contain induction vectors, at 20oC for 36 hours (L4 stage). L4 worms were harvested, washed and transferred to a new S medium supplemented with a bacteria strain HT115 expressing interfering double-strand RNA from L4440 vector under IPTG (RNAi bacteria). Worms were grown with the RNAi bacteria at 20oC until the gravid stage. Embryos were harvested from the gravid adults by hypochlorite treatment and used in experiments. In parallel, worms were fed with HT115 bacteria bearing L4440 vector without target DNA insertion (empty vector RNAi).
|
| Growth protocol |
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 |
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.
|
| Label |
Cy5
|
| Label protocol |
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 |
Input_RiIMB1_KI1967
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
sample type: input control
|
| Treatment protocol |
The N2 strain worms were arrested at the L1 larva stage in the liquid S medium for synchronization. Synchronized L1 worms were fed with a bacteria strain HB101, which do not contain induction vectors, at 20oC for 36 hours (L4 stage). L4 worms were harvested, washed and transferred to a new S medium supplemented with a bacteria strain HT115 expressing interfering double-strand RNA from L4440 vector under IPTG (RNAi bacteria). Worms were grown with the RNAi bacteria at 20oC until the gravid stage. Embryos were harvested from the gravid adults by hypochlorite treatment and used in experiments. In parallel, worms were fed with HT115 bacteria bearing L4440 vector without target DNA insertion (empty vector RNAi).
|
| Growth protocol |
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 |
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.
|
| Label |
Cy3
|
| Label protocol |
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 |
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 |
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 |
SDQ4051_LEM2_RiIMB1_KI1967_N2_MXEMB
Mixed stage N2 C. elegans embryos
|
| Data processing |
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
Genome_build: WS190
Supplementary_files_format_and_content: .wig file reports MA2C score.
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| |
|
| Submission date |
Nov 30, 2012 |
| Last update date |
Aug 15, 2016 |
| Contact name |
Kohta Ikegami |
| E-mail(s) |
kohta.ikegami@cchmc.org
|
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Department |
Division of Molecular Cardiovascular Biology
|
| Lab |
Ikegami Lab
|
| Street address |
240 Albert Sabin Way, Cincinnati, OH 45229
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL8647 |
| Series (2) |
| GSE49902 |
Chromatin organization in NPP-13 knockdown C. elegans embryos [array] |
| GSE50488 |
Chromatin organization in NPP-13 knockdown C. elegans embryos |
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