|
| Status |
Public on Sep 05, 2013 |
| Title |
IMB-1 ChIP-chip in N2 mixed-stage embryos (387661) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
SDQ4154_IMB1_EE6
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
treatment: no treatment
chip antibody: SDQ4154_IMB1 [not commercial]
|
| Treatment protocol |
For RNAi, the N2 strain worms were first 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) in the liquid S medium. 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 |
The C. elegans N2 strain animals in the liquid S-medium were fed with bacteria HB101 until becoming gravid adults. Mixed-stage embryos were harvested from the adults by hypochlorite treatment.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The C. elegans N2 strain mixed-stage embryos were cross-linked in 2% formaldehyde for 30 min at 25ºC. Chromatin extract was prepared by sonication in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl). Five to ten micrograms of antibodies were immobilized on sepharose beads and incubated with the chromatin extract for >12 hours at 4ºC. After washing the immunoprecipitants followed by RNase treatment and reverse-crosslinking, DNA were extracted and purified.
|
| 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_EE6
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
sample type: input control
|
| Treatment protocol |
For RNAi, the N2 strain worms were first 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) in the liquid S medium. 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 |
The C. elegans N2 strain animals in the liquid S-medium were fed with bacteria HB101 until becoming gravid adults. Mixed-stage embryos were harvested from the adults by hypochlorite treatment.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The C. elegans N2 strain mixed-stage embryos were cross-linked in 2% formaldehyde for 30 min at 25ºC. Chromatin extract was prepared by sonication in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl). Five to ten micrograms of antibodies were immobilized on sepharose beads and incubated with the chromatin extract for >12 hours at 4ºC. After washing the immunoprecipitants followed by RNase treatment and reverse-crosslinking, DNA were extracted and purified.
|
| 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 |
SDQ4154_IMB1_EE6_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.
|
| |
|
| Submission date |
Nov 30, 2012 |
| Last update date |
Sep 05, 2013 |
| 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) |
| GSE42651 |
Integral nuclear pore proteins bind to Pol III genes and are required for Pol III transcript processing in C. elegans [array] |
| GSE42741 |
Chromatin Immunoprecipitation of Nuclear Pore Proteins in C. elegans |
|
