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| Status |
Public on Aug 05, 2010 |
| Title |
L3-L4 hypodermal cells tiling array extraction3_array1 |
| Sample type |
RNA |
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| Source name |
L3-L4 hypodermal cells tiling array extraction3_array1 channel_1
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: NC1790 (engineered, target gene dpy-7 tagged by 3xFlag::PAB-1)
tissue: hypodermis (L3-L4)
developmental stage: L3-L4 larva 20dC 22h 23dC 24hr post-L1
genotype: unc-119(ed1); wdEx626[unc-119+; dpy-7::3xFLAG::PAB-1]
sex: Hermaphrodite
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| Growth protocol |
Animals were synchronized by treating a mixed stage hermaphrodite population with bleach to collect embryos. The embryos surviving bleach treatment were incubated without food, causing the larvae to arrest at the L1 stage upon hatching. Starved L1 larvae were plated in the presence of food and raised at 20 degrees Celsius (or indicated temperature) to desired stage.
For embryonic assays, embryonic cells were obtained using methods previously described [Christensen et al, 2002). Briefly, embryos were isolated from gravid adults following lysis in a hypochlorite solution. Intact embryos were separated from debris by flotation on 30% sucrose. For later stages, the embryos were incubated without food, causing the larvae to arrest at the L1 stage upon hatching. Starved L1 larvae were plated on standard NGM plates in the presence of food (OP50) and raised at 20 degrees Celsius. Staging was determined by examining a subset of animals under Nomarski optics at intervals for postdeirid, vulval and germline development. Animals were collected at the mid-L2, late L3-mid L4 and young adult (pre-gravid) stages and washed in M9 buffer. mid-L2 worms were collected when postdeirid divisions complete. L3/L4 animals were collected when a majority of animals diplayed a bilaterally symmetrical "christmas tree" vulval anatomy (See view at 40 hr, Fig 18, Sulston and Horvitz, Dev. Biol. 56, 110-156, 1977) Young Adult were collected when vulvae fully formed and oocytes present in gonad, but no embryos. The worms were centrifuged on a sucrose cushion to remove debris and recovered in several washes of M9 buffer.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Briefly, staged worms were treated with formaldehyde to cross link RNAs to FLAG-PAB-1, lysed with in a French pressure cell (3x for L2, 4x for L3/L4/YA), followed by glass homogenizer (30x) on ice. The lysate is cleared by centrifugation and RNA purified (trizol) from a 100 ul sample to estimate RNA (A260) in the total lysate. Homogenate (200ug total RNA) was incubated with anti-FLAG beads at 4°C for two hours and washed. The Low salt wash reduces non-specific RNA bound to the beads (see Von Stetina, Watson et al. Genome Biology 8:R135, 2007). Formaldehyde cross-linking was reversed and bound RNAs eluted by incubation at 65 C for 30 min. RNAs were extracted using Trizol/chloroform followed by isopropanol precipitation, and DNAse treatment (Column RNA cleanup from April 2006 Qiagen RNeasy Mini Handbook. RNAs were resuspended in RNase-free H2O, and stored at 20°C. The samples are polyA enriched, but there is a significant quantity of rRNA that comes down non-specifically in the prep. Note: Animals used for cell/tissue-specific profiling carry a promoter::3XFLAG::PAB-1 construct. Animals used for control samples are N2 without an 3XFLAG-tagged PAB-1, and a mockIP is performed. Therefore, the protocol is the same for cell/tissue-specific profiling and control samples, and reference samples represent background signals from the IP technique.
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| Label |
Biotin
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| Label protocol |
The NuGEN WT Pico RNA amplification kit was used to generate amplified single stranded cDNA from RNA samples. Starting material is 10 ng of RNA for mRNA tagging experiments and 1-5 ng of RNA for embryonic cells isolated by FACS. The NuGEN WT-Ovation Exon Module was used to synthesize double stranded cDNA from the amplified single stranded cDNA template. This was followed by fragmentation and labeling with the NuGEN FL-Ovation cDNA Biotin Module.
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| Hybridization protocol |
Array hybridization and processing was performed at the Vanderbilt Shared Microarray Resource (VMSR) http://array.mc.vanderbilt.edu/. Briefly, targets in Hyb cocktail were received in the VMSR for hybridization services. Affymetrix targets in hyb cocktail were heat denatured at 99C for 5', and then held at 45C for at least 5 minutes. Targets were loaded on the Affymetrix Tiling arrays and the arrays were hybridized for 16 h at 45C in a rotisserie oven. After hybridization, arrays were washed using the Affymetrix fluidics station and the Affymetrix HWS kit (cat#900720), which provides pre-made stains (SAPE and antibody) as well as final holding buffer, including wash buffers A and B. All steps were completed according to the Affymetrix technical manual. Washed arrays were scanned on an Affymetrix GeneChip Scanner 3000. Cel files were posted to the investigator.
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| Scan protocol |
Array hybridization and processing was performed at the Vanderbilt Shared Microarray Resource (VMSR) http://array.mc.vanderbilt.edu/. Briefly, targets in Hyb cocktail were received in the VMSR for hybridization services. Affymetrix targets in hyb cocktail were heat denatured at 99C for 5', and then held at 45C for at least 5 minutes. Targets were loaded on the Affymetrix Tiling arrays and the arrays were hybridized for 16 h at 45C in a rotisserie oven. After hybridization, arrays were washed using the Affymetrix fluidics station and the Affymetrix HWS kit (cat#900720), which provides pre-made stains (SAPE and antibody) as well as final holding buffer, including wash buffers A and B. All steps were completed according to the Affymetrix technical manual. Washed arrays were scanned on an Affymetrix GeneChip Scanner 3000. Cel files were posted to the investigator.
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| Description |
Antibody information listed below: official name: EZview Red ANTI-FLAG® M2 Affinity Gel;target name: FLAG;host: Mouse;antigen: DYKDDDDK;clonal: Monoclonal;purified: Affinity;company: Sigma;catalog: F-2426;reference: http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N5=Product No.|BRAND KEY&N4=F2426|SIGMA&N25=0&QS=ON&F=SPEC;short description: Binds to the FLAG epitope wherever it is located in the fusion protein: amino-terminal Met-amino-terminal carboxy-terminal or internal. Binding is not Ca2+-dependent.sSuitable for purifying N-terminal Met-N-terminal C-terminal FLAG fusion proteins 3xFLAG fusion proteins.
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| Data processing |
Tiling_Array_Signal_Extraction protocol. Mismatch (MM) probe values are subtracted from their corresponding perfect match (PM) probes. Processed data are obtained using following parameters: BPMap is BPmap:GPL5634:RW:1&oldid=22907 Tiling_Array_Normalization_and_Smoothing protocol. Biological replicates (usually 3 but sometimes 2) are normalized using quantile normalization. The normalized signal from multiple replicates are then smoothed to produce a composite signal. Each probe's value is replaced by the psuedomedian of all probes in a window around it from all replicates. This is done in a sliding window fashion shifted one probe at a time. The window size is set to 110bp, which corresponds to about 4 probes. Processed data are obtained using following parameters: window size is 110
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| Submission date |
Jul 28, 2010 |
| Last update date |
Apr 30, 2013 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
help@modencode.org
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| Phone |
416-673-8579
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| Organization name |
Ontario Institute for Cancer Research
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| Lab |
modENCODE DCC
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| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
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| Platform ID |
GPL5634 |
| Series (1) |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM571542_534DMM0224-20091012-CeT.CEL.gz |
46.1 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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