BND cellulose enriched DNA from dmc1 cells 5h after transfer to sporulation media
Treatment protocol
Meiotic single strand DNA enrichment on BND cellulose. 10 µg of genomic DNA prepared by direct phenol/chloroform extraction was digested with HindII and SspI (Roche) for 4 hours (2 units per µg of DNA), which results in a predicted median fragment size of 400 bp. 100 µl of BND cellulose slurry (corresponding to 50 µl of packed matrix) was equilibrated in TEN buffer (Tris 10 mM, EDTA 1 mM, 1M NaCl pH 8.0) by three successive centrifugations (1 min, 1000g) and resuspensions in 450 µl TEN buffer to remove particules. NaCl (5M) was added to restriction digests to 1M final, and digested material was combined with the packed BND cellulose. After 10 min incubation at room temperature with occasional mixing, matrix plus bound DNA was isolated by five successive centrifugations (1000g, 1 min) with intervening resuspension in 450 µl TEN. Single strand-enriched DNA was eluted in 5 similar washes of 450 µl of TEN + 1.7% (w/v) caffeine (Sigma). Caffeine-eluted fractions were combined, remaining BND cellulose was removed using a glass filter spin columns (Harvard Apparatus), and pooled fractions concentrated and desalted by 5 successive buffer exchanges with 500 µl TE, using a Microcon YM-30 spin-filter. Caffeine removal was monitored by measuring decrease of flow-through absorbance at 270nm against TEN buffer.
Extracted molecule
genomic DNA
Extraction protocol
Phenol/chloroform extraction
Label
Cy5
Label protocol
Two successive primer extensions (Round A) were performed on DNA samples using T7 sequenase polymerase (USB) and primerA (5'-GTTTCCCAGTCACGATCNNNNNNNNN-3'), the first 17 nt of which are absent from S. cerevisiae genome. For ssDNA-enriched material, the first extension was performed without prior denaturation to select for the single strand templates; for ChIP samples both extensions were preceded by denaturation. The following program was used: 10 °C to 37 °C ramp over 8 min; 37 °C for 8 min; 94 °C for 2 min; 10°C for 5 min while adding sequenase; 10 °C to 37 °C over 8 min; 37°C for 8 min; 24 ˚C for 10 sec. All amplifications included a second round (Round B) using Taq polymerase (Promega) and primer B (5'-GTTTCCCAGTCACGATC-3') using the following program: 95 ˚C for 30 sec; 47 ˚C for 30 sec; 72 ˚C for 2 min; for 15, 18, 21, 24 or 27 cycles. The extent of amplification was assayed by displaying 5% of each reaction on a 1.5% agarose gel and staining with SYBR green (Molecular Probes). Samples with the least number of amplification cycles showing detectable product were selected, and primer dimmers were removed by two successive filtration/wash cycles using Microcon YM-100 spin filters (Millipore) and 500 µl of TE. Aminoallyl-dUTP was then incorporated by PCR (Round C) using the same program and primers as in Round B, but a 3/2 ratio of aadUTP/dTTP and 15 units of Taq polymerase per reaction. Amplified material was purified by two successive filtration/wash cycles using Microcon YM-100 spin filters (Millipore) 500 µl of 10 mM Na2CO3 pH 8.9. 2µg of amplified material from Round C was labeled with Cy-5 monoreactive dye (GE Healthcare) and 500 ng of similarly amplified control material (BND cellulose input or whole cell extract) was labeled with Cy-3 (GE Healthcare) in 50 mM Na2CO3 pH 8.9 for 1 hour in the dark. Unincorporated dye was removed by 3 successive washes with 400 µl MES 50 mM pH 7.2 (Sigma) using Microcon YM-50 spin filters (Millipore). Dye incorporation was estimated using by measuring dye adsorbance at 550 nm (Cy-3) and 650 nm (Cy-5) in final products.
Hybridization protocol
500 ng of each labeled sample were hybridized to an Agilent yeast whole genome oligonucleotide array (Agilent, G4486A) for 17 hours at 60˚C in the 1X hybridization buffer supplied by Agilent. Slides were washed for 5 min in 6X SSPE, 0.05% (w/v) N-lauroylsarcosine, once in 0.06X SSPE for 5 minutes, and were then rinsed in the stabilizing and drying solutions supplied by Agilent.
Scan protocol
Slides were canned using an Axon 4000B scanner set at 10 µm resolution with automatically adjusting laser PMT values to achieve a maximum fluorescence saturation of 0.005%. Fluorescence data was extracted using GenePix 6.0 (Axon) software. For each channel, features were filtered according to the following criteria. First, all spots with a diameter less than 50 µm were removed. The background fluorescence in each channel was then calculated using the mean fluorescence value of 315 empty array elements. All array elements with fluorescence less than background + 2 standard deviations were removed, as were all array elements with a signal to background ratio (signal +background)/ background) less than 3.
Description
S. cerevisiae (SK1)
Data processing
Background normalization of fluorescence signals in each channel was performed using a subset of probes for which the presence of meiotic single strand DNA is unlikely. The median fluorescence intensity of background probes was used to normalize the fluorescence intensity for each spots in each channel. (see Buhler et al, Table S2 for details about the choices of background probes used in this study)
Meiotic DNA double strand breaks in the yeast Saccaromyces cerevisiae
Data table header descriptions
ID_REF
S288c specific probes
The filtering conditions described above identified a set of array elements that were consistently removed from independent hybridizations with genomic DNA, sheared by sonication to an average size of 1 kb and labeled as above with Cy-3. These elements most likely represent sequences present in the reference strain but absent from SK1. 515 of a total of 41282 array elements met this criterion and were removed from subsequent analyses. These elements are lebelled S288c
DSBs bckgrd probes
probes used for background normalization (B). Background normalization of fluorescence signals in each channel was performed using a subset of probes for which the presence of meiotic single strand DNA is unlikely. The median fluorescence intensity of background probes was used to normalize the fluorescence intensity for each spots in each channel.
