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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 09, 2023 |
| Title |
Sequencing of therapeutic transposon insertions from TT1 mouse liver [ PB5_liver] |
| Sample type |
SRA |
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| Source name |
liver
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| Organism |
Mus musculus |
| Characteristics |
tissue: liver
group: PB
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For SBE-seq whole livers of gene therapeutically treated animals were lysed in 150 ml lysis buffer (100 mM TRIS-HCl pH8, 5 mM EDTA pH8, 200 mM NaCl, 0.2% SDS) and incubated overnight at 50 °C in the presence of 300 µg/ml ProteinaseK (VWR Chemicals). 1 ml of the lysate was used for standard phenol/chloroform extraction. DNA precipitation was done with 3 volumes of absolute ethanol in the presence of 1/10 volume sodium acetate (3M). Pelleted DNA was washed with 70% ethanol, air-dryed and dissolved in nuclease-free water (SigmaAldrich).
5 μg genomic DNA from mouse liver was enzymatically fragmented, end-repaired and dA-tailed using the QIAseq FX DNA Library Kit (Qiagen). DNA was cleaned with AmpPure XP beads following each steps. Next, a linker with a special structure, disfavouring the formation of linker-linker PCR products was ligated to the genomic DNA fragments. Then the 400-1500 bp range of ligated fragments were selected using a Pippin Prep instrument (Sage Science). Next, on this selected fragment pool two nested PCRs were carried out, where one member of the PCR primer pairs was transposon-specific and the other was linker-specific. The 1st PCR was performed for 16 cycles using a biotinylated transposon-specific primer. Then the biotinylated PCR products were purified with streptavidin-coated paramagnetic beads. The 2nd PCR was performed on the purified PCR products for another 16 cycles using transposon- and linker-specific primers carrying the Illumina indexing-specific paired-end adapters to create the final NGS library. The libraries were cleaned with AmpPure XP beads. Each step of creating the NGS libraries was verified by analysing sample aliquots on a TapeStation4200 instrument (Agilent) using D1000 screen tapes. The final concentration of each library was determined by a Qubit fluorometer, and libraries were spiked with 10% PhiX control library before proceeding with Illumina run. On the Illumina instruments custom-made Read2 and Index Read primers were used instead of the standard Illumina primers. The sequencing of SBE-seq libraries was performed using an Illumina MiSeq instrument.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Paired-end reads from Illumina FASTQ files were assembled using PEAR and filtered for the presence of PB and SB ITR sequences using Smith – Watermann algorithm. Following the trimming of Illumina adapters and ITR sequences using Cutadapt reads extending at least 15 bp were considered for further analysis.
Burrows-Wheeler algorithm (BWA) was used to map trimmed reads to the Mus musculus genome version GRCm39.
To assure the quality of mapping we utilized a custom method to identify genomic regions where mismapped reads accumulate due to the imperfections of reference genome assemblies. We mapped reads from two different whole genome sequencing experiments (SRR7278720 and SRR7278736) using BWA applying the mem -a parameter. Genomic regions with 100x the average read coverage were identified. Mapped reads from our experiments residing in these identified high read coverage regions were excluded from further analysis.
The integration sites were determined as those genomic coordinates where at least 3 reads had their start point and integration site sequences were identified as the first 2 and 4 nucleotides for SB and PB transposons, respectively.
Assembly: Mus musculus genome version GRCm39.
Supplementary files format and content: PB1-17 and SB1-13 ...bed files contain the unique filtered vector integration sites identified in individual animals
Supplementary files format and content: The PB- and SBmerged_1_12 ...bed files contain the unique filtered vector integration sites merged from 1-12 animals. The number of repetitions for each recurrent integration site across the 12 experimental animals is indicated.
Library strategy: Tn-Seq
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| Submission date |
Sep 08, 2022 |
| Last update date |
Mar 09, 2023 |
| Contact name |
Lajos Mates |
| E-mail(s) |
mates.lajos@brc.hu
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| Organization name |
Biological Research Centre
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| Department |
Institute of Genetics
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| Lab |
Laboratory of Cancer Genome Research
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| Street address |
Temesvari krt. 62.
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| City |
Szeged |
| ZIP/Postal code |
H-6726 |
| Country |
Hungary |
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| Platform ID |
GPL16417 |
| Series (1) |
| GSE212895 |
Treatment of TT1 mice with transposon-based gene therapy |
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| Relations |
| BioSample |
SAMN15235909 |
| SRA |
SRX8562672 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6560672_PB5_liver_unique._integrations._filtered.bed.gz |
985.7 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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