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| Status |
Public on Feb 06, 2022 |
| Title |
MVV vector integration in sheep cells |
| Organism |
Ovis aries |
| Experiment type |
Other
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| Summary |
To test if LEDGF/p75 influences distribution of Maedi-visna virus (MVV) integration sites, we infected sheep CPT3, LKO1 (PSIP1-null), LHKO1 and LHKO2 (PSIP1/HDGFL2-null) cells with MVV-derived vector. Genomic DNA was isolated from infected cells, and chromosomal junctions at integrated U5 vDNA ends were amplified using linker-mediated PCR, sequenced using Illumina technology and mapped to sheep genome.
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| Overall design |
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 (including the 6-nucleotide barcode 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.
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| Contributor(s) |
Cherepanov P, Singh PK, Chivukula V, Engelman A |
| Citation(s) |
35504909 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R01 AI070042 |
Integrase Structural Virology |
DANA-FARBER CANCER INSTITUTE |
Alan N. Engelman |
| P50 AI150481 |
Pittsburgh Center for HIV Protein Interactions (PCHPI) |
UNIVERSITY OF PITTSBURGH AT PITTSBURGH |
ANGELA M. GRONENBORN |
| P50 GM082251 |
Pittsburgh Center for HIV Protein Interactions (PCHPI) |
UNIVERSITY OF PITTSBURGH AT PITTSBURGH |
ANGELA M. GRONENBORN |
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| Submission date |
Feb 03, 2022 |
| Last update date |
May 08, 2022 |
| Contact name |
Peter P Cherepanov |
| E-mail(s) |
Peter.Cherepanov@crick.ac.uk
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| Organization name |
The Francis Crick Institute
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| Lab |
Chromatin Structure and Mobile DNA
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| Street address |
1 Midland Road
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| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL24916 |
Illumina HiSeq 4000 (Ovis aries) |
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| Samples (4)
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| This SubSeries is part of SuperSeries: |
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| Relations |
| BioProject |
PRJNA803152 |
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