Full-length open reading frames and/or DNA binding domains were either cloned into the Gateway pDEST15 (N-terminal GST-tag) expression vector by recombinational cloning from previously created pENTR clones or were cloned by PCR amplification from genomic DNA and Gateway cloning into pDONR221. All proteins were produced from purified plasmids by in vitro transcription and translation using the PURExpress® In Vitro Protein Synthesis Kit according to manufacturer’s instructions. Glycerol was added to a final concentration of 38%, and proteins were stored at -20°C until further use. Western blots were performed for each protein to assess quality and to approximate protein concentration by visual inspection relative to a dilution series of recombinant GST standards.
Label
Alexa 488
Label protocol
Proteins were tagged with N-terminal GST by cloning. Protein-bound arrays were incubated with Alexa-488-conjugated rabbit polyclonal antibody to GST (Invitrogen).
Hybridization protocol
Double-stranded microarrays were first pre-moistened in PBS / 0.01% Triton X-100 for 5 min and blocked with PBS / 2% (wt/vol) nonfat dried milk (Sigma) under LifterSlip cover slips (Erie Scientific) for 1 h. Microarrays were then washed once with PBS / 0.1% (vol/vol) Tween-20 for 5 min and once with PBS / 0.01% Triton X-100 for 2 min. Proteins were diluted to 100 nM (unless otherwise specified) in a 175-μl protein binding reaction containing PBS / 2% (wt/vol) milk / 51.3 ng/μl salmon testes DNA (Sigma) / 0.2 μg/μl bovine serum albumin (New England Biolabs). Preincubated protein binding mixtures were applied to individual chambers of a four-chamber gasket cover slip in a steel hybridization chamber (Agilent), and the assembled microarrays were incubated for 1 h at 20°C. Microarrays were again washed once with PBS / 0.5% (vol/vol) Tween-20 for 3 min, and then once with PBS / 0.01% Triton X-100 for 2 min. Alexa-488-conjugated rabbit polyclonal antibody to GST (Invitrogen) was diluted to 50 μg/ml in PBS / 2% milk and applied to a single-chamber gasket cover slip (Agilent), and the assembled microarrays were again incubated for 1 h at 20°C. Finally, microarrays were washed twice with PBS / 0.05% (vol/vol) Tween-20 for 3 min each, and once in PBS for 2 min. Slides were spun dry by centrifugation at 40 g for 5 min. After each hour-long incubation step, microarrays and cover slips were disassembled in a staining dish filled with 500 ml of the first wash solution. All washes were performed in Coplin jars at 20°C on an orbital shaker at 125 r.p.m. Immediately following each series of washes, microarrays were rinsed in PBS (slowly removed over approximately 10 seconds) to ensure removal of detergent and uniform drying.
Scan protocol
Protein-bound microarrays were scanned to detect Alexa-488-conjugated antibody (488 nm ex, 522 nm em) using at least three different laser power settings to best capture a broad range of signal intensities and ensure signal intensities below saturation for all spots. Separately, slides were scanned after primer extension to quantify the amount of incorporated Cy3-conjugated dUTP (543 nm ex, 570 nm em). Microarray TIF images were analyzed using GenePix Pro version 6.0 software (Molecular Devices), bad spots were manually flagged and removed, and data from multiple Alexa 488 scans of the same slide were combined using masliner (MicroArray LINEar Regression) software.
Data processing
First, we normalize Alexa 488 signal by the Cy3 signal for each spot to account for differences in the total amount of double-stranded DNA. Because Cy3-dUTP incorporation is influenced both by the total number of adenines and the sequence context of each adenine, we perform a linear regression over all 41,944 variable spots to compute the relative contributions to the total signal of all trinucleotide combinations (followed by adenine). Using these regression coefficients, we calculate the ratio of observed-to-expected Cy3 intensity and use that as a normalization factor. Second, to correct for any possible non-uniformities in protein binding, we further adjust the Cy3-normalized Alexa 488 signals according to their positions on the microarray. We calculate the median normalized intensity of the 15 x 15 block centered on each spot and divide the spot’s signal by the ratio of the median within the block to the median over the entire chamber. Every non-palindromic 8-mer occurs on at least 32 spots in each chamber of our universal PBM and we provide several scores for each 8-mer in each experiment: (1) Median Intensity, (2) Z-Score, and (3) Enrichment Score (E-Score).
