protein: EGR1 protein concentration: 100nM array condition: Non-UV
Growth protocol
Cell growth protocol is detailed in the manuscript for all tested proteins and antibodies.
Extracted molecule
protein
Extraction protocol
Extract protocol is detailed in the manuscript for all tested proteins and antibodies.
Label
Alexa 488
Label protocol
Label protocol is detailed in the manuscript for all tested proteins and antibodies.
Hybridization protocol
Commercially synthesized, high-density, single-stranded DNA microarrays from Agilent were first double-stranded via primer extension as done in previous assays (Berger et al, Nature Biotechnology 2006; Shen et al, Cell Systems 2018). The arrays were partially irradiated with UVC such that only some chambers were exposed to UVC while others were not. In order to irradiate only certain parts of the array, and protect the rest from UV irradiation, a cover/gasket slide was cut to the desired size are used to partially cover the DNA array. Next, the gasket slide was fully covered using Cryo-Babies® labels, and the array-gasket slide sandwich was placed in an open container with 1x PBS. To irradiate the DNA on the array, we used a Stratalinker® UV Crosslinker 1800 instrument and conducted a series of irradiations with UVC. Using the energy mode, the container was irradiated with 100 J/m2, randomly repositioned inside the crosslinker 15 times to achieve 1,500 J/m2. Midway through the series, the PBS was replaced in order to keep the solution at room temperature. This procedure resulted in a DNA array with some chambers of the array irradiated by UV and others not irradiated. Samples with the “UV” condition used irradiated chambers while those with a “Non-UV” condition used non-irradiated chambers. The arrays were first pre-moistened in PBS / 0.01% Triton X-100 for 5 min and blocked with PBS / 2% (wt/vol) nonfat dried milk for 1 hour as done in prior assays (Shen et al, Cell Systems 2018). Proteins were incubated at the indicated concentration using the buffers referred to in the manuscript for 1 hour. 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 (Shen et al, Cell Systems 2018). Then the arrays were incubated with either Penta-His Alexa488-conjugated antibody and Penta-His Alexa647-conjugated antibody (1:20 and 1:40 ratio respectively) in a buffer with 2% (wt/vol) milk and 0.05% Tween for 1 hour (buffer details described in the manuscript). After incubation, the arrays were washed twice with PBS / 0.05% (vol/vol) Tween-20 for 3 min each, and once in PBS for 2 min as done in prior work (Shen et al, Cell Systems 2018).
Scan protocol
Protein bound microarrays were scanned at 488nm using a GenePix 4400A at multiple laser power and gain settings to best capture the signal and avoid saturation of spots. The resulting TIF images were processed using GenePix Pro 7.3.
Data processing
The raw data collected using the GenePix® 4400A microarray scanner was processed using the Seed and Wobble suite (Berger et al, Nature Biotechnology 2006), with modifications to adapt the k-mer data to our UV-Bind universal design, as described in the manuscript. For each unique sequence tested, median values over the spots containing that sequence were used unless otherwise noted (i.e. for the universal design sequences and for the competition tests). The number of replicate spots for each sequence varied between 3 and 20, depending on the DNA library. Processing of k-mers and PWMs using Seed-and-Wobble was done using the adjusted signal (i.e. Alexa488Adjusted or Alexa635Adjusted column) in the alldata.txt file as done by default (Berger et al, Nature Biotechnology 2006).
