|
| Status |
Public on Feb 06, 2022 |
| Title |
MVV vector in CPT3-LKO1 cells |
| Sample type |
SRA |
| |
|
| Source name |
CPT3-LKO1 cells infected with MVV vector
|
| Organism |
Ovis aries |
| Characteristics |
infection protocol: Cells were infected with MVV vector and processed for linker-mediated PCR 5 days post infection.
cell line: CPT3-LKO1 was generated by transfection of CPT3 with CRISPR-Cas9 RNPs targeting sheep PSIP1 gene.
|
| Growth protocol |
Cells were cultured at 37°C in 5% CO2 atmosphere in Dulbecco's modified Eagle medium (DMEM, Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotic/antimycotic solution (Sigma-Aldrich).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA isolated from cells 5 days post-infection with MVV vector was processed for linker-mediated PCR, and the amplified viral LTR-chromosome junctions were sequenced. To this end, genomic DNA, digested with MseI overnight at 37°C, was ligated to a double-stranded DNA linker containing 5'-TA overhang overnight at 12°C. Customized DNA linkers were used in conjunction with barcoded primers to aid in multiplexing and prevent crosstalk between samples. The first round and the nested MVV U5 primers were 5'-CTAATTCCGTGCAACACCG and 5'-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC TNN NNN NCA ACA CCG GAG CGG ATC, respectively (underlined sequence represents the 6 nucleotides barcode specific to LTR primer for multiplexing). The nested PCR primers contained Illumina adaptor sequences appended at the 5' ends. The PCR products, multiplexed on a single lane of a flow cell, were subjected to 150-bp paired-end sequencing on a HiSeq-4000 Illumina instrument.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Paired-end reads were cropped to remove linker and virus LTR sequences.
Cropped paired reads were aligned to sheep genome OviAri4 using BWA MEM.
High-quality alignemnts were selected with samtools view -F 4 -F 256 -q 1.
The remaining alignements were parsed to select unique integration sites and extract coordinates of the central base pair step for each site.
Genome_build: OviAri4
Supplementary_files_format_and_content: bed
|
| |
|
| Submission date |
Feb 03, 2022 |
| Last update date |
Feb 07, 2022 |
| Contact name |
Peter P Cherepanov |
| E-mail(s) |
Peter.Cherepanov@crick.ac.uk
|
| Organization name |
The Francis Crick Institute
|
| Lab |
Chromatin Structure and Mobile DNA
|
| Street address |
1 Midland Road
|
| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24916 |
| Series (2) |
|
| Relations |
| BioSample |
SAMN25635568 |
| SRA |
SRX14030926 |
