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Sample GSM5808158 Query DataSets for GSM5808158
Status Public on Mar 29, 2022
Title cd14_72PFA_b
Sample type SRA
 
Source name Primary CD14+ monocytes
Organisms Homo sapiens; Human betaherpesvirus 5
Characteristics virus strain: TB40E_GFP
cell type: CD14+ monocytes
time course: 72hpi
treatment: PFA
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
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.
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
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description cd14_hff_processed.csv
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
 
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
Organization name Weizmann Institute
Department Molecular Genetics
Lab Noam Stern-Ginossar
Street address 234 herzel
City Rehovot
ZIP/Postal code 7610001
Country Israel
 
Platform ID GPL29977
Series (1)
GSE193467 Temporal Dynamics of HCMV Gene Expression in Lytic and Latent Infections
Relations
BioSample SAMN24842267
SRA SRX13724977

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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