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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