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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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE59717 | GEO | 2015/05/26

SECONDARY ACCESSION(S): PRJNA256013

REPOSITORIES: GEO

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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.

2015-05-26 | E-GEOD-59717 | biostudies-arrayexpress

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.

2020-06-08 | GSE151912 | GEO

Total and newly transcribed (4sU-RNA) RNA of primary human fibroblasts infected with the vhs-null mutant of HSV-1

Project description:Primary human foreskin fibroblasts (HFF) were infected with vhs-null mutant of 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.

2020-10-26 | GSE129715 | GEO

4sU-seq of HFF exposed to salt and heat stress

Project description:Primary human foreskin fibroblasts (HFF) were exposed to either salt stress (80mM KCl) or heat stress (44ºC). 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.

2018-03-08 | GSE100469 | GEO

Gene expression and 4sUDRB-seq for NF90/NF110 of human scramble and KD HeLa cells.

Project description:We have optimized the metabolic tagging of newly transcribed RNAs by 4-thiouridine (4sU) to identify newly-transcribed (nascent) circRNAs from human HeLa cells.

2017-06-23 | GSE90874 | GEO

4sUDRB-seq of human PA1 cells, undifferentiated H9 cells and H9 differentiated FB neurons

Project description:We have optimized the metabolic tagging of newly transcribed RNAs by 4-thiouridine (4sU) to identify newly-transcribed (nascent) circRNAs from human PA1 cells, undifferentiated H9 cells and H9 differentiated FB neurons.

2016-04-08 | GSE73325 | GEO

4sUDRB-seq: measuring transcription elongation and initiation genomewide

Project description:A new method to measure elongation and intitiation rates Reversal inhibition of transcription with DRB and tagging newly transcribed RNA with 4-thiouridine (4sU)

2014-05-09 | E-GEOD-57116 | biostudies-arrayexpress

Explore the effect of m6A reader YTHDF1 on RNAs half-life

Project description:In order to evaluate mRNA dynamic, we performed 4SU-TT-seq to estimate RNA synthesis and degradation rates in HEK293 cell line. After 1h exposure to the nucleoside analog 4-thiouridine (4SU), 4sU is incorporated into newly transcribed RNA, and the resulting 4sU-labeled RNAs are isolated and sequenced. The sequencing results revealed that parts of mRNAs in YTHDF1 deficient cells tends to be more stable than mRNAs from wild type

2022-11-13 | GSE143994 | GEO

Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution

Project description:RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression. 4sU-tagging was performed in human DG75 B-cells by adding 500 uM 4sU to cell culture medium for 5, 10, 15, 20 or 60 min. Following isolation of total cellular RNA, this was separated into nascent and untagged, pre-existing RNA. Nascent RNA as well as total and untagged RNA from 60 min 4sU-tagging were subjected to SOLiD sequencing (SOLiD II) obtaining 35 nt reads.

2012-04-25 | E-GEOD-31653 | biostudies-arrayexpress

Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution

2012-04-26 | GSE31653 | GEO

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