transcription factor: GR protein concentration: Protein concentration: 100nM
Extracted molecule
protein
Extraction protocol
Cell growth, protein extraction, and protein labelling protocols are described in detail in the manucript, for each tested protein.
Label
Alexa 488
Label protocol
Cell growth, protein extraction, and protein labelling protocols are described in detail in the manucript, for each tested protein.
Hybridization protocol
Single-stranded DNA libraries containing transcription factor binding site sequences and all possible single base variants were commercially synthesized on microarray chips (Agilent). Double-stranded DNA was generated on the chip by hybridization with the wild-type reverse complement oligonucleotides in solution (variant complements were absent from the hybridization solution). For each wild-type sequence, the solution contained ~2.5 μM (large excess) unlabeled HPLC-purified oligonucleotides and ~0.25 μM FAM/Cy3-labelled HPLC-purified oligonucleotides (Integrated DNA Technologies). The reaction buffer mixture for the hybridization step was 100 μl 10x reaction buffer (260 mM Tris-HCl, pH 9.5, 65 mM MgCl2) in a total volume of 1000 μl, similarly to 20. The chip was incubated with reaction mixture in a hybridization oven using a pre-warmed stainless-steel chamber and gasket cover slip. After a 5-hour incubation (85 °C for 10 min, 75 °C for 10 min, 65 °C for 60 min, 60 °C for 120 min, and 55 °C for 100 min), the hybridization chamber was disassembled in a glass staining dish in 500 ml phosphate buffered saline (PBS) / 0.01% Triton X-100 at 37°C. The chip was transferred to a second staining dish, washed for 10 min in PBS / 0.01% Triton X-100 at 37°C, washed once more for 3 min in PBS at room temperature, similarly to 20. The fluorescent signal (Cy3/FAM) of hybridized oligonucleotides was measured using a GenePix 4400A microarray scanner to confirm that the hybridization was successful and reproducible, and that no significant cross-hybridization occurred. Protein binding and antibody steps were performed similarly to protein-binding microarray (PBM) assays (Berger et al, Nature Biotechnology 2006). The fluorescent signal of bound transcription factor for each DNA spot was measured using a GenePix 4400A microarray scanner.
Scan protocol
Protein-bound arrays were scanned to detect Alexa-488-conjugated or Alexa-647-conjugated (detected at 635nm wavelength) antibody 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 of interest. Microarray TIF images were analyzed using GenePix Pro version 6.0 software (Molecular Devices).
Data processing
To correct for any possible non-uniformities in protein binding, we adjusted the fluorescent signals according to their positions on the microarray. We calculated the median normalized intensity of the 15 x 15 block centered on each spot and divided the spot's signal by the ratio of the median within the block to the median over the entire chamber. For each unique sequence represented on the array, we then calculated the median over replicate spots.
