|
| Status |
Public on Apr 15, 2024 |
| Title |
NL4.3 CEMM7 n=1 5dpi |
| Sample type |
SRA |
| |
|
| Source name |
Virus-containing supernatant
|
| Organism |
Human immunodeficiency virus 1 |
| Characteristics |
tissue: Virus-containing supernatant
cell line: CEM-M7 Cas9
cell type: Cell line
|
| Treatment protocol |
For samples indicated, cells were treated with 1000U/mL of IFN-β for CEMM7 Cas9 cells and repleanished every 3 days or with 100U/mL of IFN-β for SupT1 CCR5 Cas9 cells
|
| Growth protocol |
Supernatants are collected from HIV-1 infected cell lines at various time points and after passaging of the cells. The virus in the supernantant is concentrated and lysed
|
| Extracted molecule |
other |
| Extraction protocol |
Total RNA was isolated using the Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions.
cDNA reactions were performed according to the manufacturer’s instructions of the PrimeScript™ RT Reagent Kit (Takara) using primers specifically targeting the U6 and scaffold region (forward primer 5´-CCGACTCGGTGCCACTTTTT-3´, reverse primer 5´-CGTGACGTAGAAAGTAATAATTT-CTTGGG-3´). cDNA reactions were purified using the Monarch PCR Purification Kit (NEB) and eluted in 10µl elution buffer. The gRNA cassette was amplified using the NEBNext® High-Fidelity 2X PCR Master Mix and primers including Illumina adaptors and 8nt barcodes to allow Next Generation Sequencing (NGS) analysis. PCR reactions were purified using the Monarch PCR Purification Kit (NEB) and eluted in 10µl elution buffer.
|
| |
|
| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
362_3
Counts NL4.3 in CEMM7 N=1-3.xlsx
Test NL4.3 in CEMM7 N=1-2 5 dpi untreated.xlsx
|
| Data processing |
SeqPrep to merge paired ends into one file (Using Galaxy)
FastQC for quality control (Using Galaxy)
MAGeCK count using the gRNA_list_by_gene.tab (in supplementary file) to attribute each read to a unique gRNA and a gene (Using Galaxy)
MAGeCK test comparing either one time point to input virus or dNef to WT for the same time point using the normalized counts file from the MAGeCK count (Using Galaxy)
Assembly: gRNA list are indicated in gRNA_list_by_gene.tab (in supplementary file)
Supplementary files format and content: "Counts" file are the output of the MAGeCK count showing normalized counts of the gRNA listed by genes taregtted
Supplementary files format and content: "Test" file are the genes output of the MAGeCK Test showing enrichement of the targetted genes in the condition compared to a refence condition
Supplementary files format and content: "Test sgRNA" file are the sgRNA output of the MAGeCK Test showing enrichement of the targetted genes in the condition compared to a refence condition
|
| |
|
| Submission date |
Oct 16, 2023 |
| Last update date |
Apr 15, 2024 |
| Contact name |
Konstantin MJ Sparrer |
| E-mail(s) |
Konstantin.Sparrer@uni-ulm.de
|
| Organization name |
Ulm University Medical Center
|
| Department |
Institute of Molecular Virology
|
| Street address |
Meyerhofstr. 1
|
| City |
Ulm |
| ZIP/Postal code |
89081 |
| Country |
Germany |
| |
|
| Platform ID |
GPL33848 |
| Series (1) |
| GSE245526 |
Replication-competent HIV-1 gRNA Constructs for CRISPR/Cas9-based Discovery of Antiviral Factors |
|
| Relations |
| BioSample |
SAMN37851407 |
| SRA |
SRX22108356 |
