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Status |
Public on May 05, 2023 |
Title |
attB_GN_plasmid_mix |
Sample type |
SRA |
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Source name |
None
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Organism |
synthetic construct |
Characteristics |
tissue: None cell line: E. coli cell type: Cultured cells
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Growth protocol |
DMEM with 10% FBS and 1 % Penicillin / Streptomycin
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Extracted molecule |
genomic DNA |
Extraction protocol |
HEK 293T genomic DNA was extracted using a Qiagen DNeasy Blood and Tissue Kit The DNA segments of interest were amplified from 500 ng DNA with 0.3125 μM forward and reverse primer in a 40 μL reaction with Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific, F548L) added as half the volume. Reaction conditions were 95℃ 3’, 95℃ 15”, 60℃ 15”, 72℃ 30”, repeated twenty-six to twenty-eight times, 72℃ 1’, 4℃ hold. In the case of excision via flanking GA sites, an extra forward or reverse primer was used so that both excised and unexcised products could be amplified from the same PCR tube. PCR products were run on a 2% TAE-agarose gel. DNA bands were excised and extracted using a GeneJet Gel Extraction and DNA Cleanup Micro Kit. The DNA regions of interest were sequenced by the Amplicon-EZ service provided by Genewiz (now Azenta Life Sciences), providing Illumina high throughput sequencing with read 1 and read 2 sequences of 250 nt each OTHER: Amplicon sequencing of DNA recombined within cells
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina MiSeq |
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Data processing |
The DNA regions of interest were sequenced by the Amplicon-EZ service provided by Genewiz (now Azenta Life Sciences), providing Illumina high throughput sequencing with read 1 and read 2 sequences of 250 nt each. The read 1 and read 2 fastq files from each sample were paired using PEAR v0.9.6 42. The relative frequencies of the formation of various recombined DNA products were assessed by counting the frequency of particular DNA sequences in the resulting paired fastq files using the grep function in Bash. For the excision experiments using primer set 1, excised plasmids were identified by searching for nucleotides on the right side of the attB(GA) site “ACTCCGTCGTCAGGATCATCC”, while unexcised plasmids were identified by searching for nucleotides on the right side of the attP(GA) site “ACTCAGTGGTGTACGGTACAAACC”. For the excision experiments using primer set 2, excised plasmids were identified by searching for nucleotides immediately to the left of the attP(GA) site “GATCCCCTTTAGTGAGGGTT”, while unexcised plasmids were identified by searching for nucleotides on the left of the attB(GA) site “CGACGATGTAGGTCACGGCA”. For the orthogonal GN dinucleotide experiments, the relative numbers of recombination events for the attB sequences of each central dinucleotide within the mixture was determined by searching for and counting the instances of each resulting attR sequence present in the read. For replicates 2 and 3, each sample containing a different central dinucleotide attP sequence was amplified with a unique primer marking that sample. For example, samples where the attP sequence has a central GG dinucleotide were amplified with a primer adding a G nucleotide as the first nucleotide of read 1. This allowed us to further multiplex samples, reducing overall sequencing cost. Sequence counts were imported into R, where they were converted into frequencies and plotted for the manuscript. Supplementary files format and content: "Flanked_excision_results.csv" is a comma separated value table containing the counts of each excised and unexcised amplicon for each sample and each primer set, for each replicate experiment. Supplementary files format and content: "GN_recombination_results.csv" is a comma separated value table containing the frequencies of each recombined attB sequence variant with each of the attP sequence variants, for each replicate experiment.
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Submission date |
Apr 28, 2023 |
Last update date |
May 05, 2023 |
Contact name |
Kenneth Matreyek |
E-mail(s) |
Kenneth.Matreyek@Case.edu
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Organization name |
Case Western Reserve University
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Department |
Pathology
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Lab |
Matreyek
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Street address |
2103 Cornell Rd
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City |
Cleveland |
State/province |
OH |
ZIP/Postal code |
44106 |
Country |
USA |
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Platform ID |
GPL17769 |
Series (1) |
GSE231138 |
Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants |
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Relations |
BioSample |
SAMN34429659 |
SRA |
SRX20141290 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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