that covered 89 different embryonic and adult experimental conditions and contained expression data for 13,166 probesets (corresponding to 12,229 genes in the current FlyBase release). Pearson correlation coefficients (PCC) were calculated on log2 transformed expression values, averaged for each experimental condition [14]. For each of the six prototype mitotic genes, we obtained a ranked coexpression list by calculating the PCC of the corresponding probeset with all the other probesets of the gene expression matrix. In the case of two or more probesets referring to the same gene, we considered only those showing the highest ranks. To obtain a ranked consensus co-expression list, we first scored every probeset for its presence in the upper 3000 ranks of the single co-expression lists and calculated the average PCC; we then ordered the probesets for decreasing values of these parameters (Table S1). The first 3,000 genes in this consensus co-expression list are reported in Table S1. 56 of the putative genes in the first 1000 positions of the list were either too small (less than 300 bp) or otherwise could not be amplified by PCR using at least two different pairs of primers. These putative genes (highlighted in grey in Table S1) were not assayed in our RNAi screen. Thus, to total 1000 genes, we performed RNAi for 56 additional genes that ranked from 1001 to 1056 on the consensus coexpression list (Table S1). Validation of the Coxpression Lists To ascertain the predictive value of our coexpression lists, we determined the ranks of 164 known mitotic genes in each list. These genes were selected because their ablation or knockdown by either mutation or RNAi has previously been shown to result in a strong mitotic phenotype. These 164 genes represent most of the Drosophila mitotic genes so far identified, but they do not include new mitotic genes detected in a recent RNAi screen performed by Goshima et al. [10] (see Text S1 and Table S7 for a comparison with this screen). As shown in Tables S2 and S3, 46% of these 164 genes are included in the first 1000 genes of our consensus coexpression list, which contains in total more than 13,000 genes. This non-random clustering indicates that there is both a strong tendency for mitotic genes to be transcriptionally coexpressed, as well as a strong positive correlation between the rank of a gene in the list and the probability of its involvement in mitosis. Examination of Tables S2 and S3 shows that the mitotic genes that are most tightly coexpressed are those required for proper chromosome structure and/or condensation. In contrast, the genes implicated in cytokinesis exhibit the broadest variation in expression patterns. This variation might reflect the complexity of the cytokinetic process, which requires several functions that are not specific for mitosis. For example, functions involved in regulation of the actin cytoskeleton or membrane trafficking are likely to be required in many cellular processes in addition to cell division. dsRNAs used for RNAi Recent work has raised the issue of possible off-target effects (OTEs) associated with the use of long dsRNAs for RNAi screens. It has been suggested that short homology stretches of 19 base pairs (bp) within a long dsRNA can target a gene other than the intended one [24,59,60]. In designing the primers for dsRNA synthesis, we consistently tried to obtain RNAs longer than 600 bp with a minimum content of OT sequences. The average length of the RNAs used in our screen was 750 bp; none of these RNAs carried strings of CAR triplets that are known to result in OTEs [25], but 54% of our dsRNAs contained at least one OT sequence (Table S4; OT sequences were identified using the program Drosophila Mitotic Genes developed by Flockhart et al. [61]: http://flyrnai.org/RNAi_find_ frag_free.html). However, we have several reasons to believe that very few, if any, of these OT sequences contributed to the production of the observed phenotypes. First, except for the ‘‘no dividing cells’’ (NDC) phenotype, we looked at very specific, strong and reproducible mitotic phenotypes, which are unlikely to be elicited by an OT sequence highly diluted by the excess of sequences homologous to the target gene. Second, we never found homology between any OT sequences present in the genes of the same phenocluster. Third, we found that where the data are available, the mitotic phenotypes observed in our screen match those seen in fly mutants and/or in previous RNAi experiments, regardless of whether OT sequences were present in the dsRNAs (31% of the dsRNAs that resulted in expected phenotype contained OT sequences). Finally, examination of 100 randomly chosen RNAs that did not elicit mitotic phenotypes showed that 40% of them contain OT sequences. Individual gene sequences were amplified by PCR from a pool of cDNAs obtained from 5 different libraries: 4 embryonic libraries from 0–4, 4–8, 8–12 and 12–24 hr embryos; and an imaginal disc library, all kindly provided by N. Brown [62]. If this cDNA pool did not provide the desired PCR product, DNA was amplified from genomic DNA. The primers used in the PCR reactions were 35 nt long and all contained a 59 T7 RNA polymerase binding site (59-TAATACGACTCACTATAGGGAGG-39) joined to a gene- specific sequence. The primers used to amplify the 155 mitotic genes detected in the screen are reported in Table S4. dsRNA synthesis and analysis were performed as previously described [1]. Cell cultures and RNAi Treatments S2 cells were cultured at 25uC in Shields and Sang M3 medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen). RNAi treatments were carried out according to Somma et al. [1]. 16106 cells were plated in 1 ml of serum-free medium in a well of a six-well culture dish (Sarstedt). Each culture was inoculated with 15 mg of dsRNA. After a 1 hr incubation at 25uC, 2 ml of medium supplemented with 15% FBS were added to each culture. Control cultures were prepared in the same way but without addition of dsRNA. Both RNA-treated and control cells were grown for 96 hr at 25uC, and then processed for cytological analyses. Cytological Procedures RNAi treatments were performed using six-well plates. In a typical experiment, cells in 16 wells from three plates were treated with dsRNAs, while cells in the remaining two wells served as control. After four-day incubation with dsRNA, cells from 3 ml cultures were resuspended and processed in two ways. 2 ml of this suspension were centrifuged at 800 g for 5 min, washed in 10 ml PBS, and fixed for 7 min in 3 ml 3.7% formaldehyde in PBS. Fixed cells were spun down by centrifugation, resuspended in 500 ml PBS, and cytocentrifuged onto a clean slide using a Shandon cytocentrifuge at 900 rpm for 4 min. The slides were immersed in liquid nitrogen, washed in PBS, and incubated in PBT (PBS+0.1% TritonX-100) for 15 min, and then in PBS containing 3% BSA for 20 min. These preparations were immunostained using the following antibodies, all diluted 1:100 in PBS: anti-a tubulin monoclonal DM1A (Sigma), rabbit anti- ZW10 [20], rabbit anti-cyclin B and anti-CENP-C (gifts of Christian Lehner, University of Bayreuth, Germany), and chicken anti-CID [18]. These primary antibodies were detected by incubation for 1 hr with FITC-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG (Jackson Laboratories). All slides PLoS Genetics | www.plosgenetics.org 15 July 2008 | Volume 4 | Issue 7 | e1000126