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| Status |
Public on Nov 26, 2024 |
| Title |
SupT1 infected, DisomeSeq, cyclo, 16hpi, rep2 |
| Sample type |
SRA |
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| Source name |
SupT1
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| Organisms |
Homo sapiens; Human immunodeficiency virus 1 |
| Characteristics |
cell line: SupT1
cell type: T-cell
treatment: cycloheximide
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| Treatment protocol |
For ribosome, disome and polysome profiling experiments, SupT1 cells were either mock treated (with PBS) or infected with 50 µl of virus suspension per 40 million cells by spinoculation at 1500 g for 30 min at 37 °C, in presence of 8 µg/ml polybrene (Sigma). Cells were pelleted at 1000 RPM for 3 min at 37 °C, washed once with warm media, resuspended in 40 ml RPMI medium and incubated at 37 °C.
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| Growth protocol |
SupT1 cells (NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: Sup-T1 Cells, ARP-100, contributed by Dr. Dharam Ablashi) were maintained in RPMI (Gibco) supplemented 10% FBS (Gibco), 1X L-Glutamine (Gibco), 100 μg/ml streptomycin and 100 U/ml penicillin. Cell lines were maintained at 37°C with 5% CO2.
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| Extracted molecule |
cytoplasmic RNA |
| Extraction protocol |
Ribosome profiling samples were prepared as previously described (McGlincy and Ingolia., 2017) with modifications for suspension cell lines. Briefly, at each time point of infection, cells were treated with 100 μg/ml cycloheximide in DMSO at 37 °C for 5 min. For harringtonine treated samples, cells were treated with 2 μg/ml harringtonine in DMSO at 37 °C for 5 min, followed by cycloheximide treatment. Cells were immediately pelleted at 1000 RPM for 3 min at 4 °C, washed once with cold cycloheximide containing PBS and the cell pellet was snap-frozen in liquid nitrogen and stored at -80 °C, till further use. Cell pellet was thawed in the presence of ice-cold lysis buffer (20 mM Tris-Cl pH7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml cycloheximide, 1% Triton-X, 25U/ml Turbo DNase ) and triturated 10 times through a 26G needle. The lysate was centrifuged at 17000g for 10 min at 4 °C and the supernatant was recovered. Cell lysates were subjected to RiboSeq and RNASeq.
RiboSeq libraries: RNase I footprinting of ribosomes, ribosome purification by ultracentrifugation through a sucrose cushion, RNA extraction, polyacrylamide gel size selection for footprint fragments, preadenylylated linker ligation, reverse transcription, first-strand cDNA circularization. RNASeq libraries from these samples were constructed using CORALL Total RNA-Seq Library Prep Kit (Lexogen), according to manufacturer’s instructions.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
For PRICE analysis: (1) remove adapters cutadapt -a AGATCGGAAGAGCACACG -e 0.3 (2) filter out short reads gedi -e FastqFilter -layout 2UI5U151 -min 18 (3) h.rRNA and mycoplasma removal bowtie2 -un un --local -U (4) mapping reads STAR --runMode alignReads --alignIntronMax 1 --outSAMmode NoQS --outSAMtype BAM SortedByCoordinate --alignEndsType Extend5pOfRead1 --outSAMattributes nM MD NH (5) Convert BAM to CIT gedi -e BAM2CIT -umi (6) gedi -e CorrectCIT (7) deduplicate UMIs gedi -e DedupUMI (8) gedi -e MergeCIT (9) create report gedi -e Stats (10) P-site calling gedi -e Price
cutadapt 3.5, bowtie2 version 2.3.0, STAR version=2.7.10b, Gedi version 1.0.6, Price version 1.0.4
For differential gene expression analysis: (1) Count reads per genomic feature featureCounts -O (2) normalization and log2 fold change calculations by deltaTE/DTEG.R
featureCounts version 2.0.0, deltaTE (Chothani et al. 2019)
Assembly: GCF_000001405.36, AF324493
Supplementary files format and content: The PRICE codons BED files contain the genomic regions corresponding to predicted P-sites for each RiboSeq read. The "score" column equals to the read coverage at this position.
Supplementary files format and content: The countMatrix CSV files contain the read counts overlapping each genomic feature for every time point of infection for either cyclo RiboSeq or RNASeq.
Supplementary files format and content: The deltaTE text files contain the log2 fold changes (plus statistics) for each genomic feature, normalized by DESeq2 and calculated for each timepoint of infection against the 0h mock sample. This includes RiboSeq changes, RNASeq changes and translation efficiency (TE) changes.
Supplementary files format and content: The DisomeSeq BED files contain the DisomeSeq reads that map to the HIV genome. The DisomeSeq text files contain the summed coverage per nucleotide for the HIV genome (column1: chromosome identifier; column2: genome position, column3: read coverage).
Supplementary files format and content: PRICE_report_harr_cyclo.tar contains quality control information for the RiboSeq PRICE pipeline, for both the cycloheximide and harringtonine treated samples. The index.html file allows interactive browsing through the different plots.
Supplementary files format and content: PRICE_report_disome.tar contains quality control information for the DisomeSeq PRICE pipeline. The index.html file allows interactive browsing through the different plots. Note that the p-site prediction is not valid for the DisomeSeq.
Supplementary files format and content: interactive_dTE_volcano_plots_DGE.tar contains interactive volcanoplots (translation efficiency) of the differential gene expression analysis with the deltaTE pipeline. Each infection timepoint was compared to the mock sample. Genes exceeding +/- 1 fold change and p_adj < 0.05 are colored based on consolidated gene ontology.
Supplementary files format and content: interactive_deltaTE_plots_DGE.tar contains interactive RiboSeq/RNASeq log2 fold change plots, of the differential gene expression analysis with the deltaTE pipeline. Each infection timepoint was compared to the mock sample. Only genes that are labelled as differentially expressed are plotted.
Library strategy: Ribo-seq
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| Submission date |
Oct 02, 2023 |
| Last update date |
Nov 26, 2024 |
| Contact name |
Neva Caliskan |
| E-mail(s) |
neva.caliskan@helmholtz-hiri.de
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| Phone |
+49 931 31 85298
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| Organization name |
Helmholtz Institute for RNA-based Infection Research (HIRI)
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| Lab |
Recoding Mechanisms in Infections
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| Street address |
Josef-Schneider-Str. 2/D15
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| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
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| Platform ID |
GPL33803 |
| Series (1) |
| GSE244468 |
The translational landscape of HIV-1 1 infected cells reveals novel gene regulatory principles |
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| Relations |
| BioSample |
SAMN37652022 |
| SRA |
SRX21971153 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7816493_mapped_reads_HIV_16h_disomerep2.bed.gz |
392.4 Kb |
(ftp)(http) |
BED |
| GSM7816493_read_coverage_HIV_16h_disomerep2.txt.gz |
43.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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