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Sample GSM3293889 Query DataSets for GSM3293889
Status Public on Jul 19, 2018
Title Runx1 at 200 nM concentration
Sample type protein
 
Source name Human Runx1
Organism Homo sapiens
Characteristics transcription factor: Runx1
protein concentration: Protein concentration: 200nM
Growth protocol Gateway-compatible clones containing the full-length genes for Runx1, and Runx2 were purchased from GeneCopoeia and the genes were transferred into pDEST15 using the LR Clonase reaction (Life Technologies). This vector enables the production of N-terminally GST-tagged proteins. Cells were grown in LB broth to an OD600 of 0.4 to 0.6 and then expression was induced with IPTG at 30° to 37° C. Pelleted cells were frozen, stored at -20 C, and thawed cells were lysed with lysozyme.
Extracted molecule protein
Extraction protocol GST-tagged protein was purified from the soluble portion of the lysate using GST resin (GE Healthcare) according to manufacturer’s instructions.
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) 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 200 or 100 nM 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 room temperature. 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 His (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. 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 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. 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 To correct for any possible non-uniformities in protein binding, we adjusted the Alexa 488 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. Next, the Seed-and-wobble algorithm was applied to compute enrichment scores (E-scores) and median intensities for all possible 8-mers.
Enrichment scores (E-scores), median intensities, and z-scores for 8-mers
 
Submission date Jul 19, 2018
Last update date Jul 19, 2018
Contact name Raluca Gordan
E-mail(s) raluca.gordan@duke.edu
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 GPL23935
Series (2)
GSE97794 Divergence in DNA specificity among paralogous transcription factors contributes to their differential in vivo binding
GSE117350 Divergence in DNA specificity among paralogous transcription factors contributes to their differential in vivo binding [uPBM_Runx1Runx2]

Supplementary file Size Download File type/resource
GSM3293889_Runx1_8mers_11111111.txt.gz 492.5 Kb (ftp)(http) TXT
GSM3293889_Runx1_uPBM_rawdata.txt.gz 1.1 Mb (ftp)(http) TXT
Processed data provided as supplementary file

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