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| Status |
Public on Jan 09, 2024 |
| Title |
AlphaToxin_NonClonal_U2OS_lib4 |
| Sample type |
SRA |
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| Source name |
U2OS
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| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
genotype: mutagenized U2OS cells
treatment: alpha-toxin
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| Treatment protocol |
Cells were treated twice with alpha-toxin (500 ng/ml) for 48h to ensure the selection of only highly resistant cells.
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| Growth protocol |
U2OS cells were co-transfected with piggyBac plasmid pPB-SB-CMV-puro-SD3 and p-hyPBASE using Fugene 6 transfection reagent (Promega, Madison WI, USA). For each library, between 107 - 108 cells were transfected, cultured with the addition of 3 µg/ml puromycin (Gibco) for one week to select cells that had incorporated the transposon, and then used for screens of alpha-toxin resistance with minimal further expansion. Un-mutagenized U2OS cells were treated with alpha-toxin under the same conditions to confirm that no resistance was seen in the absence of transposon mutagenesis.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
genomic DNA (gDNA) was isolated from 5-10 X 106 cells
4 mg of gDNA were digested with BfaI or CviQI (New England Biolabs, Ipswich MA, USA) and purified (QIAquick PCR purification kits, Qiagen, Hilden, Germany) then ligated with barcoded P7-side adaptors using T4 DNA Ligase (New England BioLabs). Ligated gDNA fragments were then combined and amplified by nested PCR. The first round of PCR used primers corresponding to transposon sequence (KS15) and the P7-side adaptor (LP1). A second round of PCR (primers P2A and LP22) added the full Illumina P5 and P7 sequences. Sequencing was performed by the BRI Genomics platform using a HiScan or HiSeq platform (Illumina, San Diego CA, USA) with a custom sequencing primer (Rd1SeqP.ipdC) to generate 50 or 100 bp single end reads.
described in Bruchez et al 2020
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiScanSQ |
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| Description |
combined-non-clonal.csv
lib3160_R1
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| Data processing |
Sequences were pre-processed (Trimmomatic v. 0.33) to remove Illumina adaptor sequences, filter out sequences < 40 bp and crop longer sequences to 50 bp.
Sequences were then aligned to human genome hg38 (Bowtie v. 2.1.0). Sequences that did not map to unique sites or showed significant mismatch or error (edit distance >2 over 50bp) were excluded from further analysis
Finally, reads were mapped and filtered to a known PiggyBac consensus insertion site (TTAA) at their start.
Common insertion sites (CIS) were identified using the Poisson Regression Insertion Model (PRIM) published by Bergemann et al 2012
The PRIM model was run using a p-value cutoff of 0.01 and Bonferroni correction on both selected, unselected, and in silico-generated libraries based on known TTAA sites in the genome, using window sizes of 10 kb, 25 kb, 50 kb, 75 kb, and 100 kb. CIS in experimental libraries that were also found in unselected or in silico generated libraries were excluded
Assembly: hg38
Supplementary files format and content: processed clonal data contain chromosome,mappingStart,mappingEnd,width,strand,libid,cpm,annotation,geneStart,geneEnd,geneLength,geneId,geneSymbol,normCpm,cpmRank
Supplementary files format and content: processed non clonal data contain CIS summary information and BED tracks
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| Submission date |
Sep 11, 2023 |
| Last update date |
Jan 09, 2024 |
| Contact name |
Stephanie Osmond |
| E-mail(s) |
sosmond@benaroyaresearch.org
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| Organization name |
Benaroya Research Institute
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| Street address |
1201 9th Ave
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| City |
Seattle, |
| State/province |
WA |
| ZIP/Postal code |
98101 |
| Country |
USA |
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| Platform ID |
GPL15456 |
| Series (1) |
| GSE242913 |
LITAF PROMOTES MEMBRANE REPAIR AGAINST PORE-FORMING PROTEINS |
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| Relations |
| BioSample |
SAMN37354762 |
| SRA |
SRX21751758 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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