strain: YJL8364 sample type: 3 hr re-replication induction in G2/M
Extracted molecule
genomic DNA
Extraction protocol
NaN3 was added to a final concentration of 0.1% to 250 mL cultures (arrested with nocodazole and induced with 2.7% galactose to re-replicate for 3 hr). NaN3 treated cultures were added to 25 mL of frozen, -80C, 0.2 M EDTA, 0.1% NaN3. Cells were pelleted, washed with 50 mL of 4C TE (10 mM Tris-Cl, 1 mM EDTA pH 7.5) and stored frozen at -80C. Pellets were resuspended in 4 mL lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl, 1 mM EDTA pH 8.0) and mixed with 4 mL of phenol:CHCl3:isoamyl alcohol (25:24:1) and 8 mL of acid-washed 0.5 mm glass beads (BioSpec Products, Inc., Bartlesville, OK). The suspension was vortexed seven times for 2-3 min separated by 2-3 min intervals at RT to get at least 95% of the cells lysed. The lysate was diluted with 8 mL phenol:CHCl3:isoamyl alcohol (25:24:1) and 8 mL TE, vortexed once more, and then centrifuged at 18,500 x g for 15 min at RT. After collecting the aqueous phase, the interphase was re-extracted with 8 mL TE, and the second aqueous phase from this re-extraction pooled with the first. The combined aqueous phases were extracted with an equal volume of CHCl3. The bulk of the RNA in the extract was selectively precipitated by addition of 0.01 volume 5 M NaCl (to a final concentration of 50 mM) and 0.4 volumes isopropanol followed by centrifugation at 9,000 x g for 15 min at RT. The RNA pellet was discarded and an additional 0.4 volumes of isopropanol was added to the supernatant to precipitate the DNA. Following centrifugation at 9,000 x g for 15 min at RT, the pellet was washed with 70% ethanol, dried, and resuspended with 3.5 mL of 10 mM Tris-Cl (pH 8), 1 mM EDTA. RNase A (Qiagen, Valencia, CA) was added to 340 ug/mL and the sample incubated at 37C for 30-60 min. Then Proteinase K was added to 555 ug/mL followed by another incubation at 55C for 30-60 min. Finally, 0.5 mL of 10% (w/v) Cetyltrimethylammonium Bromide (CTAB, Sigma H6269), 0.9 M NaCl (prewarmed to 65C) and 0.9 mL of 5 M NaCl was added. The sample was incubated for 20 min at 65C before being extracted with 8 mL CHCl3:isoamyl alcohol (24:1) and centrifuged at 6000 x g for 15-180 min at RT. The DNA in the aqueous phase was precipitated with 0.8 volumes isopropanol at RT, washed with 70% ethanol, dried, and resuspended in 6 mL of 25 mM Tris-Cl (pH 7), 1 mM EDTA. RNase A (Qiagen, Valencia, CA) was added to 33 ug/mL and the sample incubated at 37C for 15 min. Then the following were added to the sample in the order listed: 1) 1.5 mL of 5 M NaCl; 2) 0.5 mL of 1M MOPS (pH 7); 3) 0.5 mL of Triton X-100 (3% vol/vol); 4) 1.5 mL of isopropanol. The sample was then mixed by vortexing, then purified on a Qiagen Genomic-tip 100/G column as per the manufacturer's instructions (Qiagen, Valencia, CA). The eluted DNA was precipitated with 0.8 volumes isopropanol at 4C, washed with 70% ethanol, dried, and resuspended in 275 uL of 2 mM Tris-Cl pH 7.8. Genomic DNA was then sheared by sonication with a Branson Sonifier 450 to an average fragment size of 500 bp.
Label
Cy5
Label protocol
amino-allyl-dUTP incorporation with Klenow. Chemical coupling of dye to aa-dUTP labelled DNA.
strain: YJL7695 sample type: reference DNA (G2/M arrested with nocodazole)
Extracted molecule
genomic DNA
Extraction protocol
NaN3 was added to a final concentration of 0.1% to 250 mL or 450 mL cultures that were arrested with nocodazole (final concentration = 15 ug/mL). NaN3 treated cultures were added to 25 mL or 50 mL (for 250 mL or 450 mL cultures, respectively) of frozen, -80C, 0.2 M EDTA, 0.1% NaN3. Cells were pelleted, washed with 50 mL of 4C TE (10 mM Tris-Cl, 1 mM EDTA pH 7.5) and stored frozen at -80C. Pellets were resuspended in 4 mL lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl, 1 mM EDTA pH 8.0) and mixed with 4 mL of phenol:CHCl3:isoamyl alcohol (25:24:1) and 8 mL of acid-washed 0.5 mm glass beads (BioSpec Products, Inc., Bartlesville, OK). The suspension was vortexed seven times for 2-3 min separated by 2-3 min intervals at RT to get at least 95% of the cells lysed. The lysate was diluted with 8 mL phenol:CHCl3:isoamyl alcohol (25:24:1) and 8 mL TE, vortexed once more, and then centrifuged at 18,500 x g for 15 min at RT. After collecting the aqueous phase, the interphase was re-extracted with 8 mL TE, and the second aqueous phase from this re-extraction pooled with the first. The combined aqueous phases were extracted with an equal volume of CHCl3. The bulk of the RNA in the extract was selectively precipitated by addition of 0.01 volume 5 M NaCl (to a final concentration of 50 mM) and 0.4 volumes isopropanol followed by centrifugation at 9,000 x g for 15 min at RT. The RNA pellet was discarded and an additional 0.4 volumes of isopropanol was added to the supernatant to precipitate the DNA. Following centrifugation at 9,000 x g for 15 min at RT, the pellet was washed with 70% ethanol, dried, and resuspended with 3.5 mL of 10 mM Tris-Cl (pH 8), 1 mM EDTA. RNase A (Qiagen, Valencia, CA) was added to 340 ug/mL and the sample incubated at 37C for 30-60 min. Then Proteinase K was added to 555 ug/mL followed by another incubation at 55C for 30-60 min. Finally, 0.5 mL of 10% (w/v) Cetyltrimethylammonium Bromide (CTAB, Sigma H6269), 0.9 M NaCl (prewarmed to 65C) and 0.9 mL of 5 M NaCl was added. The sample was incubated for 20 min at 65C before being extracted with 8 mL CHCl3:isoamyl alcohol (24:1) and centrifuged at 6000 x g for 15-180 min at RT. The DNA in the aqueous phase was precipitated with 0.8 volumes isopropanol at RT, washed with 70% ethanol, dried, and resuspended in 6 mL of 25 mM Tris-Cl (pH 7), 1 mM EDTA. RNase A (Qiagen, Valencia, CA) was added to 33 ug/mL and the sample incubated at 37C for 15 min. Then the following were added to the sample in the order listed: 1) 1.5 mL of 5 M NaCl; 2) 0.5 mL of 1M MOPS (pH 7); 3) 0.5 mL of Triton X-100 (3% vol/vol); 4) 1.5 mL of isopropanol. The sample was then mixed by vortexing, then purified on a Qiagen Genomic-tip 100/G column as per the manufacturer's instructions (Qiagen, Valencia, CA). The eluted DNA was precipitated with 0.8 volumes isopropanol at 4C, washed with 70% ethanol, dried, and resuspended in 275 uL of 2 mM Tris-Cl pH 7.8. Genomic DNA was then sheared by sonication with a Branson Sonifier 450 to an average fragment size of 500 bp.
Label
Cy3
Label protocol
amino-allyl-dUTP incorporation with Klenow. Chemical coupling of dye to aa-dUTP labelled DNA.
Hybridization protocol
Hybridization was performed in 3X SSC + 25mM HEPES pH7.0 + 0.25% SDS at 63C for 18-48 hours.
Scan protocol
Arrays were scanned with an Axon 3000B scanner using GenePix4.0 or GenePixPro6.0.
Description
Figure S4
Data processing
Data were filtered in GenePix to flag as bad features that had : (1) obvious defects; (2) saturated pixels; (3) regression r^2 values less than 0.5; (4) fewer than 55% of their pixels with flourescence intensity greater than 2 standard deviations above background. BLAST analysis was used to generate a filter that would flag as bad elements whose PCR product were excessively repetitive. Raw Cy5/Cy3 median of ratios values were normalized such that the average ratio was equal to 2. VALUE is the log2 of the normalized ratio of Cy5 to Cy3; note that to convert to usable copy number profiles raise two to the indicated value.
Single-Stranded Annealing Induced by Re-Initiation of Replication Origins Provides a Novel and Efficient Mechanism for Generating Copy Number Expansion via Non-Allelic Homologous Recombination.
Data table header descriptions
ID_REF
VALUE
Value is the log2 of the normalized ratio of Cy5 to Cy3
