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Sample GSM2327870

Query DataSets for GSM2327870

Status

Public on Dec 05, 2016

Title

Sox2R75EOct4_coopseq (POU bound)

Sample type

SRA

Source name

Synthesized DNA

Organism

synthetic construct

Characteristics

library: DNA target oligos
transcription factor(s): Sox2R75EOct4

Extracted molecule

other

Extraction protocol

All EMSA were done with Sox and Oct proteins 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 following oligos GAGTCGTCTCGTCAGCACCGGCCATTGTNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGCATTGTNNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGCATTGTNGNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCCATTGTNGGNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTNGGCNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTNGGCGNTGCTAATGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTNGGCGGNTGCTAATACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTNGGCGGCNTGCTAATCCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGCATTGTTATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGCATTGTTNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCCATTGTTNNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTTNGNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTTNGGNATGCTAATGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGCATTGTTATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGCATTGTTNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCCATTGTTNNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTTNGNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTTNGGNATTAGCATGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGAACAATGATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGAACAATGNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCAACAATGNNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACAACAATGNGNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACAACAATGNGGNATGCTAATGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGAACAATGATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGAACAATGNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCAACAATGNNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACAACAATGNGNATTAGCATAGACCGTAGAGCACTCAGGTC, and GAGTCGTCTCGTCAGCACAACAATGNGGNATTAGCATGACCGTAGAGCACTCAGGTC. DNA library was designed by flanking the degenerate sequences of interest with sequences GAGTCGTCTCGTCAGCAC and CCGTAGAGCACTCAGGTC 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.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Description

Contains monomer bound for downstream processing. Contains raw data from 2 replicates.
Processed data file: coopseq_Chang_allData.txt

Data processing

Library strategy: Coop-seq
Extracting binding sites sequence from raw reads. Each read follows the format of GAGTCGTCTCGTCAGCAC-binding site-CCGTAGAGCACTCAGGTC.
Count read numbers for each variant, classified by dimer bound, Sox bound, POU bound, or unbound in coopseq 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.
For coopseq, the cooperativity, omega, is calculated by (Dimer bound x unbound)/(Sox bound x POU bound).
Supplementary_files_format_and_content: Tab delimited text files. See Processed_data_description.txt.

Submission date

Sep 25, 2016

Last update date

May 15, 2019

Contact name

Gary D. Stormo

E-mail(s)

[email protected]

Phone

(314) 362-2722

Organization name

Washington University School of Medicine