Tiling Arrays for mRNA Display Figure 1. Scheme of iterative screening for protein interactions using the IVV method and a tiling array. A cDNA library was transcribed to an mRNA library, and then ligated with PEG Puro spacer [p(dCp)2-T(Fluor)p-PEGp-(dCp)2-puromycin] using T4 RNA ligase to prepare an IVV library. In this study, bait protein fused with tandem affinity purification (TAP) tag was co-translated with the IVV library in a wheat germ extract cell-free translation system. Complexes of bait protein and target IVV molecules were captured with affinity beads (IgG agarose) via the added TAP-tag. The RNA portion of the captured IVVs was reverse-transcribed and PCR-amplified. Finally, screened cDNA libraries were labeled and hybridized with tiling arrays as an alternative to cloning and sequencing (conventional IVV method). doi:10.1371/journal.pone.0001646.g001 Design and construction of the TFT array Oligonucleotide arrays were constructed photolithographically by an oligo DNA microarray construction service (NimbleGen). The sequences of 1,562 mouse transcription regulatory factors listed by Gunji et al. [23], as well as 37 Jun-associated protein candidates found in our previous studies [14,21,22], were collected from the RefSeq (http://www.ncbi.nlm.nih.gov/RefSeq/) and Genbank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = Nucleotide) databases. Both strands of the total of 1,599 mRNA sequences were doubly represented by a total of 334,372 oligo DNA probes 50-mer in length, with no gap between the probes (Figure 1). Sample labeling, hybridization and signal detection Biotin-labeling of the samples was performed by means of in vitro transcription from an SP6 promoter at the 59-end of each cDNA fragment in the libraries, as described [24], with some modifica- tions. In this process, biotin-labeled sense-strand RNA fragments were produced. Thus, only the antisense-strand probe set was further analyzed in this study. The labeled samples from the bait (+) and bait (2) screening were hybridized separately on the tiling arrays. The hybridized tiling arrays were stained with Cy3- Streptavidin (Amersham) and detection was done with a scanner. Details are given in Methods S1 online. Data analysis Collected data from the tiling array were normalized with the median correction algorithm. Ratios of signal values between the two samples from bait (+) and bait (2) screenings were calculated (Data S1 online). The ratio data were expressed as log2X (X is the actual measurement). After signal measurement, specific signal peaks were identified by the ‘‘Windowed Threshold Detection’’ algorithm in SignalMap software (NimbleGen). This algorithm looks for at least four data points that are above a threshold value within a window. These points were grouped together and presented as a peak. We used the following parameters in the algorithm: Peak Window Size, 300 bp; Percent of Peak Threshold, 20% of maximum data in each mRNA sequence. The value of each peak was the maximum value of the data points in that peak. Only reproducible peaks in the duplicated data were collected as candidates for Jun interactors (Table S1 online). A probe set for NM_183316 was not analyzed, because the sequence of NM_183316 overlapped with that of NM_025925 on the array. Real-time PCR analysis Real-time PCR was performed with SYBR Premix Ex Taq (Takara) and protein-specific primer sets (Table S2 online) on the LightCycler (Roche) as previously described [22]. The standard PLoS ONE | www.plosone.org 2 February 2008 | Volume 3 | Issue 2 | e1646