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Sample GSM6938263 Query DataSets for GSM6938263
Status Public on Jan 20, 2023
Title 64PP_UV_r2
Sample type other
Source name Mouse Anti-6-4PP
Organism Mus musculus
Characteristics antibody: Anti-6-4 Photoproduct
antibody ratio: 1:80
array condition: UV
Growth protocol Cell growth protocol is detailed in the manuscript for all tested proteins and antibodies.
Extracted molecule other
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). Antibodies for CPD and 6-4PP were incubated in a 1:400 and 1:80 ratio respectively in a 1x PBS buffer of 2% (wt/vol) milk and 0.05% Tween for 1 hour. 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). Then the arrays were incubated with a secondary antibody of conjugated anti-mouse 488 at 1.3% volume in a PBS buffer with 2% (wt/vol) milk and 0.05% Tween for 1 hour. The arrays were then washed as previously described.
Scan protocol Anti-CPD and anti-6-4PP 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.
Description Antibody
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).
Submission date Jan 17, 2023
Last update date Jan 20, 2023
Contact name Raluca Gordan
Organization name Duke University
Department Center for Genomic and Computational Biology
Street address 101 Science Dr, CIEMAS 2179
City Durham
State/province NC
ZIP/Postal code 27708
Country USA
Platform ID GPL33026
Series (2)
GSE223012 UV irradiation remodels the specificity landscape of transcription factors [84702_PhotoProduct_Antibody]
GSE223023 UV irradiation remodels the specificity landscape of transcription factors

Data table header descriptions
VALUE Signal intensity

Data table
Ctrl_UUV_000001 3024
Ctrl_UUV_000002 12795
Ctrl_UUV_000003 5710
Ctrl_UUV_000004 8874
Ctrl_UUV_000005 11939
Ctrl_UUV_000006 5344
Ctrl_UUV_000007 9313
Ctrl_UUV_000008 4026
Ctrl_UUV_000009 5169
Ctrl_UUV_000010 14212
Ctrl_UUV_000011 20394
Ctrl_UUV_000012 3842
Ctrl_UUV_000013 7959
Ctrl_UUV_000014 14796
Ctrl_UUV_000015 15449
Ctrl_UUV_000016 6362
Ctrl_UUV_000017 7157
Ctrl_UUV_000018 4840
Ctrl_UUV_000019 3068
Ctrl_UUV_000020 15445

Total number of rows: 175866

Table truncated, full table size 3651 Kbytes.

Supplementary file Size Download File type/resource
GSM6938263_64PP_UV_r2_84702_alldata.txt.gz 6.4 Mb (ftp)(http) TXT
Processed data included within Sample table

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