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Status |
Public on Oct 11, 2017 |
Title |
BATF-IRF8-Specseq-unbound |
Sample type |
SRA |
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Source name |
Synthesized DNA
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Organism |
synthetic construct |
Characteristics |
library: DNA oligos
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Extracted molecule |
other |
Extraction protocol |
All EMSA were done in 1X NEB Cutsmart buffer (50mM Potassium Acetate, 20mM Tris-acetate, 10mM Magnesium Acetate, 100μg/ml BSA, pH 7.9@25°C) supplemented with 10% glycerol with 1mM of dsDNA and were incubated for 30 minutes on ice. Electrophoresis mobility assay (EMSA) were done using native 12% PAGE prepared as Tris/Glycine (25mM Tris pH 8.3; 192mM glycine) mini-gels. These gels were first pre-run using 1X Tris/Glycine buffer (25mM Tris pH 8.3; 192mM glycine) at 200V for 30 minutes, then samples were loaded for an additional run of 60 minutes at 200 V at 4°C. After EMSA, the gels were stained with ethidium bromide and visualized using Biorad gel imager. Each band detected in the EMSA were excised with a plastic tool and the DNA in the gel extracted by incubating for 30 mins at 50°C in 50ml acrylamide gel extraction buffer (500mM Ammonium acetate, 10mM Magnesium Acetate, 1mM EDTA, 0.1% SDS). 0.01 pmole of PCR Control DNA with the sequence of GAGTCGTCTCGTCAGCACCGGCGGCGGTTCCCGGAAAGACCGTAGAGCACTCAGGTC (primer binding sites underlined) were added to each extracted sample. Sample in the extraction buffer were purified with QIAquick Nucleotide Removal Kit (Qiagen) following the manufacturer’s instructions. Each fraction of DNA was barcoded and amplified using HotStart PCR Master Mix (Lambda Biotech). DNA was denatured at 94°C for 30 seconds, annealed at 55°C for 30 seconds and extend at 72°C for 45 seconds per round for 12 to 20 rounds with modified Indexed-Illumina primers (PE1-Genetics1/2, PE2.0). The PCR product was then purified again using QIAquick Nucleotide Removal Kit. DNA library was constructed by mixing the degenerate sequences of interest. DNA library was designed by flanking the degenerate sequences of interest with sequences GAGTCGTCTCGTCAGCAC and CCGTAGAGCACTCAGGTC or GATAGTCTCATTTTCACC and TTCCATTACAGTATCTGT for downstream processing. To make double-stranded DNA (dsDNA) libraries, 100 pmole single-strand degenerate template sequences were mixed with an equal amount of reverse complement primer (GACCTGAGTGCTCTACGG). In the presence of Taq Polymerase, brief 10-sec denaturing followed by 10 min of 55 degree annealing/extension is sufficient to make dsDNA libraries. Because any unextended single-stranded DNA (ssDNA) could contaminate the unbound band, the reaction mix was digested by 1 ml NEB Exo I exo- nuclease (New England Biolabs, Beverly, MA) for 30 min. All final dsDNA products were purified by QIAGEN (Valencia, CA) PCR purification columns.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina MiSeq |
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Description |
Conatins unbound reads needed to calculate the relative binding energy. BATF-IRF8-Specseq_processed.txt
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Data processing |
Extracting binding sites sequence from raw reads. Each read follows the format of GAGTCGTCTCGTCAGCAC-binding site-CCGTAGAGCACTCAGGTC for samples 1 to 6 and follows the format of GATAGTCTCATTTTCACC-binding site-TTCCATTACAGTATCTGT for samples 7 to 15. Count read numbers for each variant and classfied by bound or unbound in specseq. For specseq, calculate the bound/unbound ratio for each variant, then rank variants according to their relative affinities. Genome_build: N/A Supplementary_files_format_and_content: Tab delimited text files.
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Submission date |
Aug 03, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Gary D. Stormo |
E-mail(s) |
stormo@genetics.wustl.edu
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Phone |
(314) 362-2722
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Organization name |
Washington University School of Medicine
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Department |
Genetics
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Lab |
Stormo
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Street address |
4515 McKinley Ave. Room 4208.
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City |
St. Louis |
State/province |
MO |
ZIP/Postal code |
63110 |
Country |
USA |
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Platform ID |
GPL17769 |
Series (1) |
GSE102219 |
Quantitative profiling of BATF family proteins/JUNB/IRF hetero-trimers using Spec-seq |
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Relations |
BioSample |
SAMN07447591 |
SRA |
SRX3058593 |
Supplementary data files not provided |
SRA Run Selector![Help](/coreweb/images/long_help4.gif) |
Raw data are available in SRA |
Processed data are available on Series record |
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