at 30uC in a water bath for 10 min, and incubated at 37uC for the times indicated on Figure 4. Cells were immediately harvested by centrifugation, re-suspended in 700 ml of TES buffer (10 mM Tris- HCl (pH 7.4), 10 mM EDTA and 0.5% SDS), snap frozen in liquid nitrogen and stored at 280uC. The experiment was conducted on BY and RM simultaneously, and repeated four times at different days. Total RNA was extracted using the following procedure: 700 ml of room temperature phenol was added to the cell extract, mixed well by vortexing and incubated at 65uC for 20 minutes. Extract was then snap frozen in liquid nitrogen for 1 min, thawed at room temperature, centrifuged at 13000 rpm for 5 min and the upper aqueous phase was transferred to a fresh microcentrifuge tube. Once again, 700 ml of room temperature phenol was added, mixed well and centrifuged at 13000 rpm for 5 min. The upper aqueous phase was transferred to a fresh tube, 700 ml of chloroform was added, mixed well and centrifuged at 13000 rpm for 5 min, the upper aqueous phase was purified using RNeasy mini kit (Qiagen) and eluted in 50 ml nuclease free water. RNA was precipitated by adding 50 ml of 3 M NaAc, and 1.25 ml of ice-cold ethyl alcohol to the purified samples followed by incubation at 220uC for 30 min. RNA was pelleted by centrifugation at 13000 rpm for 5 min, washed once with ice-cold 70% ethyl alcohol at 13000 rpm for 5 min and re-suspended in 50 ml of nuclease water. RNA concentration was quantified based on spectral absorbance using a NanoDrop ND-1000 Spectrophotometer. Reverse transcription and real time quantitative PCR were performed on a Stratagene MX3000P real-time PCR system using the Superscript III Platinum SYBR Green One-Step qRT-PCR kit from Invitrogen following manufacturer’s protocol. Primers were 59-GTCT GTTTCGTCCATTGAAGG-39 and 59- GTCCTTAGAGTCC ACGTGTCC-39 for AHA1, 59-ATGGATTCTGAGGTTGCT GC- 39 and 59-TGGGAAGACAGCACGAGGAG-39 for ACT1, 59-GATGCAAAGAGATTAGAAACAGCG -39 and 59-GCCTT CCAACTCCTTTTGTCTA -39 for SSA3, 59-GATGAAGAA CTACGTGCTGCTG-39 and 59-GCTTCGCAGACCATTGT CG-39 for FES1 and 59-CATTCCTTCTATCCATGGCC-39 and 59-GCTTCCCGTCCAAATGAG-39 for CPR6. Amplification efficiencies and relative quantification of AHA1/ACT1, SSA3/ ACT1, FES1/ACT1 and CPR6/ACT1 ratios were calculated as described by Pfaffl [50]. Genome alignment Genome sequences of S288c (isogenic to BY) and RM were downloaded in December 2007 from NCBI (ftp://ftp.ncbi.nih. gov/genomes/Saccharomyces_cerevisiae) and the Broad Institute (http://www.broad.mit.edu/annotation/genome/saccharomyces_ cerevisiae/Home.html), respectively. The RM genome 8X assembly originates from whole genome shotgun and consists of 17 high- quality supercontigs (hqSC hence after) totalizing 11.7 Mb. The17 hqSC of RM were aligned on the 16 nuclear chromosome sequences of BY by using the nucmer algorithm implemented in MUMmer version 3.0 [51] with options –maxgap = 1000 –mincluster = 50 considering BY chromosomes as the references and RM hqSCs as the queries. The output of nucmer was then filtered and formatted using the delta-filter and show-coords programs of the MUMmer package. At this stage, the output of our alignment pipeline consisted on a list of clusters of perfect matches between regions of BY chromosomes and RM hqSCs. We implemented an automatic rule to relate RM hqSCs to BY chromosomes by maximizing the coverage and alignment quality chromosome by chromosome. We then dynamically resolved overlapping clusters of perfect matches in order to get the longest aligned fragments of RM hqSCs along each BY chromosome. Visual inspections were also used in a few cases in Single Nucleosome Epi-Polymorphisms order to define optimal boundaries of alignments. Detailed results of this genome alignment process as well as the hybrid shell/perl script used to do the genome alignment are available upon request. Sequence polymorphisms Polymorphisms between BY and RM were detected by base substitution in the final alignment. Since base calling information were not available for RM sequences, we assumed that quality was reasonable and uniform along the RM genome sequence. From the 54,039 polymorphisms found, a few targeted repeated sequences (in both genomes as annotated by RepeatMasker (open- 3.1.9, Smit, AFA, Hubley, R & Green, P. RepeatMaskerOpen-3.0. 1996–2004 http://www.repeatmasker.org) and were thus exclud- ed, leading to a final core set of 52,280 polymorphisms consisting in 47,011 SNPs (90%), 2,448 insertions (4.5%) and 2,821 deletions (5.5%). These 2,448 insertions corresponded to 238,087 bp that were absent in RM while the 2,821 deletions corresponded to 80,349 bp absent in BY. This discrepancy between BY and RM insertions is mainly due to the heterogeneous content of Ty elements between both genomes (see below). Gene prediction and comparison BY gene annotations were extracted from chromosomal features defined at NCBI website (ftp://genome-ftp.stanford.edu/pub/yeast/ chromosomal_feature/saccharomyces_cerevisiae.gff). For RM, pre- dicted gene set was downloaded from the Broad Institute website (http://www.broad.mit.edu/annotation/genome/saccharomyces_ cerevisiae/Downloads.html). which were obtained using a combi- nation of mapped ORFs from SGD predictions (http://www. yeastgenome.org), Glimmer [52] and GeneMark [53]. More details on this automated gene prediction pipeline are posted at http:// www.broad.mit.edu/annotation/genome/saccharomyces_cerevisiae/ GeneFinding.html#prediction. This original annotation file consisted of 5695 gene loci. 83 were dubious and discarded, as their annotated coding sequence did not code for a protein. 80 other predictions were also discarded because they fell in unaligned regions and could not be mapped to BY. Orthologs were identified using Blastp from WUBlast version 2.0 (Gish, W. (1996–2004) http://blast.wustl.edu) by aligning protein sequences of the 5532 remaining RM genes against the protein sequences of 6608 BY genes, and selecting reciprocal matches that fulfilled all following criteria: i) E-value ,1.e-5, ii) percentage of identity .40% and iii) match length .75% of both protein sequences. This way, 5200 RM genes were called orthologs of BY genes, and 328 genes were RM-specific. Transcript boundaries Transcript boundaries for BY genes were obtained from the complete published set of experimentally detected transcripts [23,54] available at http://www.ebi.ac.uk/huber-srv/actinomycinD. Only transcript segments overlapping .50% of a non-dubious annotated coding region on the 59 end were retained. This resulted in 4,714 verified transcription segments. We then mapped the BY transcript boundaries of ortholog genes on the RM genome, leading to 4,612 transcription segments on RM. Ty elements We extracted the DNA sequences of the 50 active Ty elements annotated in the BY genome (as defined in the NCBI chromosomal features, leading to 31 Ty1, 13 Ty2, 2 Ty3, 3 Ty4 and 1 Ty5) and blasted them onto the 17 RM supercontigs. We found 10 matches on RM with 10 Ty2 elements (percentage of identity .90% and match length .95%). No match was found with any other Ty elements even after relaxing these criteria, PLoS Genetics | www.plosgenetics.org 9 April 2010 | Volume 6 | Issue 4 | e1000913