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Sample GSM7844538 Query DataSets for GSM7844538
Status Public on Apr 15, 2024
Title NL4.3 CEMM7 n=3 10dpi
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_21
Counts NL4.3 in CEMM7 N=1-3.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 SAMN37851389
SRA SRX22108389

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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