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Status |
Public on Mar 29, 2022 |
Title |
cd14_72PFA_b |
Sample type |
SRA |
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Source name |
Primary CD14+ monocytes
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Organisms |
Homo sapiens; Human betaherpesvirus 5 |
Characteristics |
virus strain: TB40E_GFP cell type: CD14+ monocytes time course: 72hpi treatment: PFA
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Treatment protocol |
For the time course analysis fibroblasts and CD14+ monocytes were infected with the same stock of HCMV strain TB40E-GFP at a multiplicity of infection (MOI) of 1 and 10, respectively. For all other experiments CD14+ monocytes or THP1 cells were infected with HCMV strain TB40E-GFP at an MOI of 5. Cells were incubated with the virus for 2h, washed, and supplemented with fresh medium. CHX was added to infected fibroblasts and CD14+ monocytes immediately after infection at a final concentration of 100 ug/ml and 200 ug/ml or at 100 ug/ml, respectively. PFA was added to fibroblasts and CD14+ monocytes immediately after infection at a final concentration of 400 ug/ml. for epigenetic drug treatments - 48 hpi, TB40-infected CD14+ monocytes were divided into 1.2 ml tubes containing ~100,000 cells per tube and inhibitors or DMSO for negative control were added to a final concentration of 1uM for 24 hours
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Growth protocol |
Human foreskin fibroblasts (ATCC CRL-1634) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/ml penicillin and streptomycin (Beit-Haemek, Israel) and maintained at 37°C in a 5% CO2 incubator. Primary CD14+ monocytes were isolated from fresh venous blood, obtained from healthy donors, using a Lymphoprep (StemCell Technologies) density gradient followed by magnetically activated cell sorting with CD14 magnetic beads (Miltenyi Biotec). CD14+ cells were cultured in X-Vivo 15 medium (Lonza) supplemented with 2.25 mM L-glutamine and maintained at 37°C in a 5% CO2 incubator.
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Extracted molecule |
total RNA |
Extraction protocol |
for cd14 and hff time course experiment as well as TSA time course RNA was extracted using Tri-Reagent (Sigma-Aldrich), total RNA was extracted. poly-A selection was performed using Dynabeads mRNA DIRECT. For MARseq libraries epigenetic treatments RNA was extracted directly from cells using Dynabeads mRNA DIRECT. Briefly, mRNA samples of ~4ng were subjected to DNaseI treatment and 3′ dephosphorylation using FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and T4 PNK (NEB) followed by 3′ adaptor ligation using T4 ligase (NEB). The ligated products were used for reverse transcription with SSIII (Invitrogen) for first-strand cDNA synthesis. The cDNA products were 3′ ligated with a second adaptor using T4 ligase and amplified with 8 cycles of PCR for final library products of 200–300 base pairs. For epigenetic drug-treated CD14+ monocyte samples, RNA libraries were generated from samples of ~100,000 cells according to the MARS-seq protocol
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
cd14_hff_processed.csv
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Data processing |
For the time course libraries, raw sequences were first trimmed at their 3′ end, removing the illumina adapter and polyA tail. Alignment was performed using Bowtie (allowing up to 2 mismatches) and reads were aligned to concatenation of the human (hg19) and the viral genomes (NCBI EF999921.1). Reads aligned to ribosomal RNA were removed. Reads that were not aligned to the genome were then aligned to the transcriptome. Analysis of libraries generated with the MARS-seq protocol was done as previously described . Briefly, 37-bp reads were aligned using Bowtie (allowing up to 2 mismatches) to concatenation of the human (hg19) and the viral genomes (NCBI EF999921.1). Counting of reads per gene was done based on unique molecular identifiers (UMIs) (8 bp). The transcription units of the virus were based on NCBI annotations, with some changes, including merging several transcripts (considering that the library maps only the 3’ ends of transcripts) and adding some antisense transcripts. Genome_build: human (hg19) and the viral genomes (NCBI EF999921.1) Supplementary_files_format_and_content: All processed files are comma delimited files with the number of reads on the transcripts
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Submission date |
Jan 11, 2022 |
Last update date |
Mar 29, 2022 |
Contact name |
Noam Stern-Ginossar |
E-mail(s) |
noam.stern-ginossar@weizmann.ac.il
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Organization name |
Weizmann Institute
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Department |
Molecular Genetics
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Lab |
Noam Stern-Ginossar
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Street address |
234 herzel
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City |
Rehovot |
ZIP/Postal code |
7610001 |
Country |
Israel |
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Platform ID |
GPL29977 |
Series (1) |
GSE193467 |
Temporal Dynamics of HCMV Gene Expression in Lytic and Latent Infections |
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Relations |
BioSample |
SAMN24842267 |
SRA |
SRX13724977 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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