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| Status |
Public on Aug 05, 2010 |
| Title |
pathogen Smarcescens 25dC 48hr exposure post-adulthood N2 tiling array extraction2_array1 |
| Sample type |
RNA |
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| Source name |
pathogen Smarcescens 25dC 48hr exposure post-adulthood N2 tiling array extraction2_array1 channel_1
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
developmental stage: Young Adult (pre-gravid) 25dC 46 hrs post-L1
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 20/25 degrees Celsius (as specified).
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 25 degrees Celsius. Staging was determined by examining a subset of animals under Nomarski optics at intervals for vulval and germline development. Animals were collected at the mid-L2, mid-L3, mid-L4, and young adult (pre-gravid) stages and washed in S basal buffer. Exact times for growth after plating of starved L1s (all at 25 degrees C) were as follows: (1) L1 worms were grown for 4 hours (2) L2 worms were grown for 17.75 hours (Pn.p cells visible but not divided, gonad just starting to proliferate); (3) L3 for 26.75 hours (Pn.p cells divided once or twice; gonad just starting to turn up); (4) L4 for 34.25 hours (vulvae are in Christmas tree stage gonad has passed bend, sperm are present); and (5) Young Adult for 46 hours (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 S basal.
Bacterial strains included E. coli OP50, E. faecalis OG1RF, S. marcescens Db11, and P. luminescens Hb. Liquid cultures of E. coli, P. luminescens and S. marcescens were grown in LB, E. faecalis in BHI. We spread 50-150 ul of overnight bacterial liquid culture (concentrated 10-fold), depending on size of the assay plate (35 or 90 mm diameter), onto fresh NGM agar plates and incubated them for 24 h. P. luminescens were cultured at 30°C; E. coli, S. marcescens and E. faecalis at 37°C. We used 90 mm plates.Aliquots of synchronized larvae were transferred after 16-20 h to NGM plates spread with OP50 and cultivated at 25°C until just reaching adulthood. Worms were then transferred to assay plates. After 24 h or 48 h at 25°C, the worms were collected, washed three times in M9 buffer and pelleted and suspended in 10 volumes of Trizol.(Samples received as worms-in-trizol from Jonathan Ewbank's lab.)
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation from medium- to large-scale preparations of staged C. elegans nematodes. The worms were centrifuged on a sucrose cushion to remove debris and recovered in several washes of S basal. Total RNA isolation was performed by adding 4 volumes of Trizol (Gibco) per volume of packed worms. After two rounds of freeze/thaw and vortexing, the slurry was extracted with 2 volumes of chloroform. The aqueous layer was precipitated with one volume of isopropanol and the pellet washed with 70% ethanol. The pellet was resuspended in water and the concentration determined by UV spectrometry. RNA quality was assessed by examining an aliquot run on an ethidium bromide stained gel for discrete ribosomal and transfer RNA bands, as well as an mRNA "smear".
RNA is DNase treated as follows: using the Ambion DNA-free kit, add 5ul of 10x DNAse I buffer and 1ul of DNAse I to 10ug total RNA in 50ul volume. Incubate at 37deg C for 30'. Add 5ul DNase inactivation reagent and mix well. Incubate 2-3' at RT. Spin 1.5' at 10K. Put supernatant in clean RNAase-free tube. Measure concentration by spectrophotometer. We follow the standard Affymetrix protocol for cDNA synthesis, using the GeneChip® WT Double-Stranded cDNA Synthesis Kit (#900813). Everything is according to manufacturer.
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| Label |
biotin
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| Label protocol |
Array hybridization and processing performed at the Keck Facility at Yale University. Briefly, array hybridization buffer (100 mM MES, 1M [Na+], 20 mM EDTA, 0.01 % Tween 20) is used to prehybridize the array for 10-15 minutes at 450C. The prehybridized solution is removed and replaced with 80 ul of hybridization mixture containing hybridization buffer, fragmented cRNA (0.05ìg/ìl) and herring sperm DNA (0.1 mg/ml; Promega). Also included in the hybridization buffer are acetylated BSA (0.5 mg/ml, Invitrogen) and four control bacterial and phage cRNA (1.5 pM BioB, 5 pM Bio C, 25 pM BioD, and 100 pM Cre) samples to serve as internal controls for hybridization efficiency. The arrays are hybridized for 16 h at 450 in a rotisserie oven. After hybridization, arrays are washed using the Affymetrix fluidics station and stained with streptavidin-phycoerythrin (10 ìg/ml, Molecular Probes) according to the Affymetrix technical manual. Washed arrays will be scanned on a Affymetrix GeneChip Scanner 3000. Scanned output files will be visually inspected for hybridization artifacts and then analyzed by using Affymetrix GeneChip Operating Software (GCOS).
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| Hybridization protocol |
Array hybridization and processing performed at the Keck Facility at Yale University. Briefly, array hybridization buffer (100 mM MES, 1M [Na+], 20 mM EDTA, 0.01 % Tween 20) is used to prehybridize the array for 10-15 minutes at 450C. The prehybridized solution is removed and replaced with 80 ul of hybridization mixture containing hybridization buffer, fragmented cRNA (0.05ìg/ìl) and herring sperm DNA (0.1 mg/ml; Promega). Also included in the hybridization buffer are acetylated BSA (0.5 mg/ml, Invitrogen) and four control bacterial and phage cRNA (1.5 pM BioB, 5 pM Bio C, 25 pM BioD, and 100 pM Cre) samples to serve as internal controls for hybridization efficiency. The arrays are hybridized for 16 h at 450 in a rotisserie oven. After hybridization, arrays are washed using the Affymetrix fluidics station and stained with streptavidin-phycoerythrin (10 ìg/ml, Molecular Probes) according to the Affymetrix technical manual. Washed arrays will be scanned on a Affymetrix GeneChip Scanner 3000. Scanned output files will be visually inspected for hybridization artifacts and then analyzed by using Affymetrix GeneChip Operating Software (GCOS).
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| Scan protocol |
Array hybridization and processing performed at the Keck Facility at Yale University. Briefly, array hybridization buffer (100 mM MES, 1M [Na+], 20 mM EDTA, 0.01 % Tween 20) is used to prehybridize the array for 10-15 minutes at 450C. The prehybridized solution is removed and replaced with 80 ul of hybridization mixture containing hybridization buffer, fragmented cRNA (0.05ìg/ìl) and herring sperm DNA (0.1 mg/ml; Promega). Also included in the hybridization buffer are acetylated BSA (0.5 mg/ml, Invitrogen) and four control bacterial and phage cRNA (1.5 pM BioB, 5 pM Bio C, 25 pM BioD, and 100 pM Cre) samples to serve as internal controls for hybridization efficiency. The arrays are hybridized for 16 h at 450 in a rotisserie oven. After hybridization, arrays are washed using the Affymetrix fluidics station and stained with streptavidin-phycoerythrin (10 ìg/ml, Molecular Probes) according to the Affymetrix technical manual. Washed arrays will be scanned on a Affymetrix GeneChip Scanner 3000. Scanned output files will be visually inspected for hybridization artifacts and then analyzed by using Affymetrix GeneChip Operating Software (GCOS).
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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) |
| GSE23274 |
pathogen_Smarcescens_25dC_48hr_exposure_post-adulthood_N2_tiling_array |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM571502_JYOU_CTIL_Db-3-48_011309_Ce25b_MR_v02.CEL.gz |
45.8 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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