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Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA from different virus strains and mutant viruses

ABSTRACT: Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA from different virus strains and mutant viruses

PROVIDER: PRJNA637636 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11937720_1.fastq.gz	Fastqsanger.gz
	SRR11937720_2.fastq.gz	Fastqsanger.gz
	SRR11937721_1.fastq.gz	Fastqsanger.gz
	SRR11937721_2.fastq.gz	Fastqsanger.gz
	SRR11937722_1.fastq.gz	Fastqsanger.gz

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Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA

Project description:Primary human foreskin fibroblasts (HFF) were infected with wild-type simplex virus 1 (HSV-1) strain 17 at a multiplicity of infection (MOI) of 10. Newly transcribed RNA was labelled by adding 500µM 4-thiouridine (4sU) to the cell culture media for 1h. Total cellular RNA was isolated using Trizol. Newly transcribed RNA was purified following the protocol described in Raedle et al. JoVE 2013. Newly transcribed RNA was labelled in one hour intervals during the first eight hours of HSV-1 infection. All nine 4sU-RNA samples as well as total cellular RNA of every second hour of infection were sent for sequencing. Two independent biological replicates were analysed.

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Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA

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Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA from different virus strains and mutant viruses

Project description:Primary human foreskin fibroblasts (HFF) were infected with the indicated HSV-1 strains and mutants thereof at a multiplicity of infection (MOI) of 10. Newly transcribed RNA was labelled by adding 500µM 4-thiouridine (4sU) to the cell culture media for 1h. Total cellular RNA was isolated using Trizol. Newly transcribed RNA was purified following the protocol described in Raedle et al. JoVE 2013.

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Project description:Primary human foreskin fibroblasts (HFF) were infected with wild-type simplex virus 1 (HSV-1) strain 17 at a multiplicity of infection (MOI) of 10. Ribosome profiling was performed at various times during infection with minor modification to the protocol described in Stern-Ginossar N et al., Science 2012 Ribosome profiling was performed a 0, 1, 2, 4, 6 and 8 h post infection. Two biological replicates were analysed.

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Project description:Noncoding variants play a central role in the genetics of complex traits, but we still lack a full description of the main molecular pathways through which they act. Here we used molecular data to quantify the contribution of cis-acting genetic effects at each major stage of gene regulation from chromatin to proteins, within a population sample of Yoruba lymphoblastoid cell lines (LCLs). We performed 4sU metabolic labeled transcripts in 65 YRI LCLs to identify genetic variants that affect transcription rates. As expected, we found an important contribution of genetic variation via chromatin, contributing â¼65% of eQTLs (expression Quantitative Trait Loci). The remaining eQTLs, which are not asso- ciated with chromatin-level variation, are highly enriched in transcribed regions, and hence may affect expression through co- or post-transcriptional processes. International HapMap lymphoblastoid cell lines (LCLs) derived from YRI (Yoruba in Ibadan, Nigeria); We adapted the 4sU labelling method from (PMID 21516085). Briefly, cell cultures were grown to log phase in volumes sufficient to yield about 300 ng of 4sU-labeled RNA. Cells were incubated with 4sU for the required length of time (0, 30, or 60 minutes), then washed, pelleted, and frozen. Total RNA was extracted, and 4sU-labeled RNA was separated from total RNA using a bead-based biotin-streptavidin purification protocol. We sequenced metabolic labeled transcripts in 65 YRI LCLs 30 minutes and 60 minutes after incubation.

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