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| Status |
Public on Apr 29, 2022 |
| Title |
B-LGP2-HIV1-1 |
| Sample type |
SRA |
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| Source name |
OUTPUT RNA extracted on LGP2 protein
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| Organisms |
Homo sapiens; Human immunodeficiency virus 1 |
| Characteristics |
cell line: 4X4 HEK293
genotype/variation: LGP2 protein tagged 1-STrEP tag
infection: HIV-1
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| Treatment protocol |
ST-RLR4X4 (tagged) cells were co-cultivated with HIV-1-infected cells MT4C5 cells .5*107 MT4C5 (donor cells) were exposed 150ng (equivalent p24) of HIV-1 NL4.3 for 2hr at 37°C. After washing the virus, the cells were grown for 48h. The infection was monitored by flow cytometry analysis by intracellular Gag staining. Infection was then performed via coculture of ST-RLR4X4 cells and MT4C5 cells at a donor:target cell ratio of 1:1. 24 h post infection, cells were lysed and affinity purification of ST-tagged proteins and RNA extraction was performed as described in DOI: 10.7554/eLife.11275
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| Growth protocol |
Cells were maintained in DMEM-Glutamax (GIBCO, Thermo Fisher Scientific, Waltham, Massachusetts) supplemented with 10% heat-inactivated FCS (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts),100 U/ml/100 mg/ml of Penicillin-Streptomycin (GIBCO), and G418 at 500 mg/ml (#G8168, SIGMA, St. Louis, Missouri).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed twice with cold PBS and lysed in 6 mL of lysis buffer (20 mM MOPS-KOH [pH 7.4], 120 mM KCl, 0.5% Igepal, 2 mM b-mercaptoethanol), supplemented with 200 U/mL RNasin (Promega) and Complete Protease Inhibitor Cocktail (Roche). Cell lysates were incubated on ice for 20 min, with gentle mixing every 5 min, and then clarified by centrifugation at 16,0003g for 15 min at 4°C. A 250-mL aliquot of each cell lysate was used to perform total RNA purification using TRI Reagent LS [Sigma]). The remaining cell lysate was incubated for 2 hr on a spinning wheel at 4°C with 200 mL StrepTactin Sepharose High Performance beads (GE Healthcare). Beads were collected by centrifugation (1,600 g) for 5 min at 4°C) and washed twice for 5 min on a spinning wheel with 5 mL washing buffer (20mMMOPS-KOH [pH 7.4], 120mMKCl, 2mMb-mercaptoethanol) supplemented with 200 U/mL RNasin and complete protease inhibitor cocktail. Precipitates were eluted using biotin elution buffer (IBA Lifesciences). RNA purification was performed using TRI Reagent LS (T3934; Sigma). RNA was dissolved in 50 mL DNase-free and RNase-free ultrapure water. Extracted RNAs were analyzed using Nanovue (GE Healthcare) and the Bioanalyser RNA nano kit (Agilent Technologies).
Before RNA-seq analysis of total and RLR-bound RNA from virus- and mock-infected cells, depletion of ribosomal RNA was done using the riboZero reagents included in the TruSeq stranded total RNA library prep kit (#20020596, Illumina). NGS libraries were generated following the manufacturer’s protocol. The indexed samples were multiplexed per 4 or 6 and sequenced on a HiSeq2500 sequencer (Illumina) to produce single-ends 65 bases reads, bearing strand specificity.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
B-LGP2-1_GCCAAT_L007
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| Data processing |
Reads were cleaned of adapter sequences and low-quality sequences using cutadapt version 1.11.Only sequences at least 25 nt in length were considered for further analysis
Bowtie version 1.2.2 (parameters --chunkmbs 400 -m 1) was used for alignment on the reference genome.
Genes were counted using featureCounts version 1.4.6-p3 from the Subreads package (parameters: -t gene -g ID -s 1).
To compute coverage on viruses, Bowtie version 1.2.2 14 , with default parameters, was used for alignment on the reference genomes. Only first base of each read are used as input of genomeCoverageBed from Bedtools 15 . Minus and plus strand coverage are separated by a simple awk command.
For statistical analysis of NGS data, count data were analyzed using R version 3.5.1 and the Bioconductor package DESeq2 version 1.20.0 16 . The normalization and dispersion estimation were performed with DESeq2 using the default parameters and statistical tests for differential expression were performed applying the independent filtering algorithm. For each virus, a generalized linear model including the replicate, beads and protein factors as well as the beads x protein interaction was set in order to test for the differential expression between the biological conditions. For each pairwise comparison, raw p-values were adjusted for multiple testing according to the Benjamini and Hochberg (BH) procedure 17 and genes with an adjusted p-value lower than 0.05 were considered differentially expressed.
Supplementary_files_format_and_content: Matrix count for each sample (after genomeCoverageBed)
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| Submission date |
Jul 25, 2019 |
| Last update date |
Apr 29, 2022 |
| Contact name |
Rachel Legendre |
| E-mail(s) |
rachel.legendre@pasteur.fr
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| Organization name |
Institut Pasteur
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| Department |
Research and Resource Center for Scientific Informatics
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| Lab |
Hub of Bioinformatics and Biostatistics
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| Street address |
28, rue du docteur Roux
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| City |
Paris |
| ZIP/Postal code |
75724 |
| Country |
France |
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| Platform ID |
GPL26693 |
| Series (2) |
| GSE134857 |
Viral Mimicry by Endogenous Y-RNAs Contributes to Antiviral Immunity [HIV] |
| GSE134861 |
Viral Mimicry by Endogenous Y-RNAs Contributes to Antiviral Immunity |
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| Relations |
| BioSample |
SAMN12361543 |
| SRA |
SRX6589511 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3972807_B-LGP2-1_HIVNL43_count.txt.gz |
29.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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