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| Status |
Public on Aug 05, 2010 |
| Title |
embryo all cells reference tiling array extraction1_array1 |
| Sample type |
RNA |
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| Source name |
embryo all cells reference tiling array extraction1_array1 channel_1
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
tissue: reference (embryo)
developmental stage: Mixed stage of embryos 20dC
genotype: wild type
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.
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. Eggshells were removed by incubation in 0.5 ml chitinase (0.5 U/ml in egg buffer) for 45 minutes. Following resuspension in L-15 medium supplemented with 10% FBS (L15-10) and antibiotics, the embryos were dissociated by passage through a 5?m syringe filter (Durapore). Cells were plated on poly-L-lysine (0.01%, Sigma) coated single-well chambered coverglasses (Nalgene Nunc International) at a density of ~10 million cells/ml and maintained in L15-10 media. Cells were incubated at 25°C in a humidified chamber. Wildtype (N2) cells were isolated and treated similarly.FACSSorting experiments were performed on a FACStar Plus flow cytometer equipped with a 488 nm argon laser or on a FACS Aria (488nm, 633 nm lasers) (Becton Dickinson, San Jose, CA). Emission filters were 530 ± 30 nm for GFP fluorescence and 585 ± 22 nm for PI fluorescence. The machine was flushed with egg buffer [6] prior to sorting to enhance viability. 2 um fluorescent beads were used to calibrate light scattering parameters for the relatively small size of C. elegans embryonic cells. Cells were sorted at a rate of 4000-5000 cells per second through a 70 um nozzle. Immediately prior to sorting, supernatant from the 24 hour cultures was removed and discarded. 1 ml of egg buffer was added to the chamber coverglass. Cells are loosely adherent to poly-L-lysine and can be easily dislodged with gentle pipetting. 3 ml of egg buffer + cells were drawn into a 3 cc syringe and the suspension filtered with a 5 ?m Durapore syringe filter. Propidium Iodide (PI) was added to the cell suspension at a final concentration of 5 ug/ml prior to sorting. Autofluorescence levels were established by flow cytometry of cells isolated from wildtype (i.e. non-GFP) embryos (See Fig 2A). Next, wildtype cells stained with PI were used to define the sorting gate for damaged cells. GFP+ cells containing no PI were sorted to establish the intensity range of GFP fluorescence. Finally, unc-4::GFP cells stained with PI were gated (Fig 2B) using the parameters established above. The sorting gate for size and granularity (Fig 2C) was empirically adjusted to exclude cell clumps and debris and to achieve ~90% enrichment for GFP-labeled cells. unc-4::GFP cells were collected in a 15 ml conical tube containing 1 ml of L15-10 media. Cells were pelleted using low-speed cenrifugation (300 × g) and either plated on peanut lectincoated slides for visualization [6] or used for RNA isolation (see below). Reference cells were obtained from 1 day old cultures of embryonic blastomeres isolated from the non-GFP wildtype strain (N2). In this case, all viable cells (i.e. non-PI stained) were collected by FACS for RNA isolation.(from Fox et al.,2005)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Add cells lysate/Trizol (from FACS) to Phase Lock Gel Heavy tubes, extract with phenol/chloroform and precipitate RNA with isopropanol. Wash pellet with 75% ethanol and dissolve pellet in DEPC dwater for purification and concentration with ZYMO DNA-free RNA kit(Detailed protocol: MAPCeL_RNA_isolation.doc)
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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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| 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 |
Aug 05, 2010 |
| 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) |
| GSE23246 |
embryo all cells reference tiling array |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM571419_534DMM58-20080118-CeT.CEL.gz |
39.9 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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