population: Ancestral generations: 0 individual: 1 sample: final
Treatment protocol
This is the ancestral control line. It was cryogenically frozen at −86°C throughout the MA period of the other lines.
Growth protocol
The population was grown on NGM plates (Nematode Growth Medium) at 20˚C and maintained on 90x15 mm petri dishes seeded with 750μl of Escherichia coli (OP50) in YT medium.
Extracted molecule
genomic DNA
Extraction protocol
DNA was prepared either by standard phenol-chloroform extraction followed by ethanol precipitation, or with the Puregene DNA Purification Kit (D-7000A, Gentra Systems) using solid tissue protocol.
Label
Cy5
Label protocol
Cy3/Cy5 dye-labeled random 9mers (TriLink BioTechnologies, Inc.) were diluted to 1 O.D./42 μl of buffer containing 0.125M Tris-HCl (pH 8.0), 0.125M MgCl2, 1.75 μl/ml β-Mercaptoethanol. 1 μg of genomic DNA was added to each random 9mer buffer solution, denatured at 95 °C and then chilled on ice in 0.2 ml PCR tubes. 10 μl of 50X dNTP mixture (1X TE buffer, 10 mM each of dATP, dCTP, dGTP, and dTTP), 8 μl DI water and 100 U Klenow fragment (exo-) was added to each tube and mixede well with a pipet. Samples were centrifuged and incubated at 37 °C for 2 hours. 10 μl 0.5 M EDTA was added and mixed well to stop the labeling reaction. DNA was precpitated by adding 11.5 μl 0.5 M NaCl and 110 μl isopropanol, vortexing, incubating in the dark for 10 minutes at room temperature, and centrfuging at 12,000X G for 10 minutes. The supernatant was removed and the DNA pellet was washed with 500 μl 80% ethanol. After centrifugation at 12,000X G for 2 minutes, the supernatant was removed and the pellet was dried in a SpeedVac on low heat for 5 minutes before being rehydrated in 25 μl DI water. DNA concentration was measured using a spectrophotometer. See Maydan JS, Lorch A, Edgley ML, Flibotte S, and Moerman DG. 2010. Copy number variation in the genomes of twelve natural isolates of Caenorhabditis elegans. BMC Genomics Jan 25;11:62 (PMID: 20100350)
population: 100A generations: 409 individual: 2 sample: final
Treatment protocol
Spontaneous mutation accumulation via population bottlenecks of N = 100 every generation.
Growth protocol
The population was grown on NGM plates (Nematode Growth Medium) at 20˚C and maintained on 90x15 mm petri dishes seeded with 750μl of Escherichia coli (OP50) in YT medium.
Extracted molecule
genomic DNA
Extraction protocol
DNA was prepared either by standard phenol-chloroform extraction followed by ethanol precipitation, or with the Puregene DNA Purification Kit (D-7000A, Gentra Systems) using solid tissue protocol.
Label
Cy3
Label protocol
Cy3/Cy5 dye-labeled random 9mers (TriLink BioTechnologies, Inc.) were diluted to 1 O.D./42 μl of buffer containing 0.125M Tris-HCl (pH 8.0), 0.125M MgCl2, 1.75 μl/ml β-Mercaptoethanol. 1 μg of genomic DNA was added to each random 9mer buffer solution, denatured at 95 °C and then chilled on ice in 0.2 ml PCR tubes. 10 μl of 50X dNTP mixture (1X TE buffer, 10 mM each of dATP, dCTP, dGTP, and dTTP), 8 μl DI water and 100 U Klenow fragment (exo-) was added to each tube and mixede well with a pipet. Samples were centrifuged and incubated at 37 °C for 2 hours. 10 μl 0.5 M EDTA was added and mixed well to stop the labeling reaction. DNA was precpitated by adding 11.5 μl 0.5 M NaCl and 110 μl isopropanol, vortexing, incubating in the dark for 10 minutes at room temperature, and centrfuging at 12,000X G for 10 minutes. The supernatant was removed and the DNA pellet was washed with 500 μl 80% ethanol. After centrifugation at 12,000X G for 2 minutes, the supernatant was removed and the pellet was dried in a SpeedVac on low heat for 5 minutes before being rehydrated in 25 μl DI water. DNA concentration was measured using a spectrophotometer. See Maydan JS, Lorch A, Edgley ML, Flibotte S, and Moerman DG. 2010. Copy number variation in the genomes of twelve natural isolates of Caenorhabditis elegans. BMC Genomics Jan 25;11:62 (PMID: 20100350)
Hybridization protocol
The sample was rehydrated in 40 μl of NimbleGen Hybridization Buffer (NimbleGen Systems, Inc.), denatured at 95 °C for 5 min, and then cooled to 42 °C. Hybridizations were carried out for 18 hr at 42 °C in the NimbleGen Service Laboratory. The arrays were washed using a NimbleGen Wash Buffer System (NimbleGen Systems, Inc.) and immediately dried down by centrifugation. See Selzer,R.R., T.A. Richmond, N.J. Pofahl, R.D. Green, P.S. Eis, P. Nair, A.R. Brothman, and R.L. Stallings. 2005. Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44:305-319 (PMID 16075461).
Scan protocol
The scanner used was an Axon Scanner (Model # 40008). See Selzer,R.R., T.A. Richmond, N.J. Pofahl, R.D. Green, P.S. Eis, P. Nair, A.R. Brothman, and R.L. Stallings. 2005. Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44:305-319 (PMID 16075461).
Description
Genome variation profiling by genome tiling array
Data processing
The raw log2 intensity ratios were normalized with the LOESS regression.
