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| Status |
Public on May 05, 2014 |
| Title |
EMSA-Seq of p50p50 round 2 of SELEX |
| Sample type |
SRA |
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| Source name |
p50p50
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| Organism |
Homo sapiens |
| Characteristics |
selex round: 2
nf-kb dimer: p50p50
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each round of SELEX selection, two protein binding reactions (all 10 μl) were performed simultaneously. Reaction 1 contained 200 ng 64-bp positive dsDNA, 115 ng (Reaction 2) p50 protein (Promega) in 10 mM Tris-HCl, pH7.5, 50 mM NaCl, 0.5 mM EDTA, 3 mM MgCl2, 1 % glycerol, 0.5 mg/ml BSA, 0.05% NP-40, 0.01 mM DTT. Reaction 2 contained 200 ng 64-bp random dsDNA, 115 ng p50 protein in 10 mM Tris-HCl, pH7.5, 50 mM NaCl, 0.5 mM EDTA, 3 mM MgCl2, 1 % glycerol, 0.5 mg/ml BSA, 0.05% NP-40, 0.01 mM DTT and 0.1 μg polydI-dC (Amersham). The protein binding reactions were incubated at room temperature for 60 min and loaded on 6% native polyacrylamide gel and migrated in 0.5 × TBE buffer for 1.5 hs. After electrophoresis, the gel was directly visualized on UV transilluminator and the gel slice containing the shifted dsDNA (Reaction 2) was excised according to the position of the shifted band of Reaction 1. The gel slice was socked in 100 μl diffusion buffer [0.5 M NH4Ac, 10 mM Mg(Ac)2, 1 mM EDTA, pH8.0, 0.1% SDS] overnight at 37°C. The diffusion buffer was purified with Microcon YM-30 and DNA was eluted in 25 μl ddH2O. For experiments in which several rounds of SELEX were carried out the following procedure was adopted. DNA obtained after the first round of TF-DNA binding followed by EMSA, elution and purification was amplified using a high-fidelity PCR procedure. Briefly, multiple 10 μl reactions each comprised of 1 mM dNTPs, 4μM of Primers 1 and 2 (using nested primers: R-F1 and R-R1 for Round 1, R-F2 and R-R2 for Round 2; R-F3 and R-R3 for Round 3; R-F4 and R-R4 for Round 4), together with 1 units of PrimerStar® HS Taq polymerase (TaKaRa) buffered in accompanying 1X PCR Buffer were incubated for 3min at 94°C, then subjected to 20 PCR cycles (1min 94°C, 1min 60°C, 2 min 70°C). DNA was pooled, purified with Microcon YM-30 (Millipore). This was subsequently used as starting material for TF-DNA binding in the second round of SELEX.
The dsDNAs selected in Round 0 to 4 were pooled in equal molar to form a multiplexed sample for sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
EMSA-Seq selection of ligands that bind a transcription factor
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| Data processing |
Basecalls performed using CASAVA version 1.4
Raw data in the form of reads (FASTQ format) obtained after deep sequencing has been subsequently processed using both UNIX and perl scripts. The main processes included (i)trimming the adaptor sequence from each reads; (ii) matching the sequences of the paired reads to remove the reads with unmatcghed sequences; (iii) checking the definite base calling (A, C,G and T) to remove reads with N; (iv) checking the valid barcode and constant regions to remove reads with invallid barcode and constant regions including incorrect bases and length; (v)checking the unique random sequence at the length of 16 bp to remove reads with incorrect length; (vi)sorting reads according to barcodes: the reads belonging to Round 4 to 0 were ended with AAAA and TTTT, GAAG and GTTT, AGAG and GTCT, AAAG and GTGT, and CAAG and GTGC, respectively; (vii) removing flanking constant regions and barcode sequences from reads and obtained the 16-mer target sequences.
Genome_build: No genome build was perfomed. This item is inapplicable to this study.
Supplementary_files_format_and_content: Fasta files for the final 16-mer sequences obtained for Round 0 to 4.
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| Submission date |
Jul 10, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jinke Wang |
| E-mail(s) |
wangjinke@seu.edu.cn
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| Phone |
86-25-83793620
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| Organization name |
Southeast Uiniversity
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| Department |
Biomedicine
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| Lab |
Wang's Lab
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| Street address |
Sipailou 2
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| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
210096 |
| Country |
China |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE48660 |
An improved SELEX-Seq strategy for characterizing DNA-binding specificity of transcription factor: NF-κB as an example |
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| Relations |
| BioSample |
SAMN02230338 |
| SRA |
SRX319493 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1183033_EMSA-Seq_of_p50p50_round_2_of_SELEX-16mer_sequences.fa.gz |
15.7 Mb |
(ftp)(http) |
FA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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