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Transcription profiling of human B-cells (BL41) for tc-, nt- and p-RNA half-life determination

ABSTRACT: RNA was labeled in BL41 cells by culturing cells for 60 min in media containing 100ÂµM 4sU. Tc-RNA was separated into nt- and p-RNA. All three RNA subsets were subjected to microarray analysis. Only probe sets providing present calls in all RNA samples/subsets were included into the analysis; We used microarrays to determine half-lives of mRNAs expressed in BL41 cells Experiment Overall Design: RNA was labeled in BL41 cells by culturing cells for 60 min in media containing 100ÂµM 4sU. Tc-RNA was separated into nt- and p-RNA. All three RNA subsets were subjected to microarray analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Lars DÃ¶lken

PROVIDER: E-GEOD-9973 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Half-life determination for human B-cells (BL41)

Project description:RNA was labeled in BL41 cells by culturing cells for 60 min in media containing 100µM 4sU. Tc-RNA was separated into nt- and p-RNA. All three RNA subsets were subjected to microarray analysis. Only probe sets providing present calls in all RNA samples/subsets were included into the analysis We used microarrays to determine half-lives of mRNAs expressed in BL41 cells Keywords: Determination of mRNA half-life in human B-cells

2009-09-30 | GSE9973 | GEO

Transcription profiling of mouse NIH-3T3 cells for tc-, nt- and p-RNA half-life determination

Project description:Data from tc-, nt- and p-RNA as well as 1 and 2h of actinomycin-D treatment (5Âµg/ml) of NIH-3T3 cells used to determine half-lives. RNA was labeled for 15, 30 or 60 minutes with 4-thiouridine. After preparation of tc-RNA, thiol-labeled RNA was biotinylated using biot-HPDP and subsequently tc-RNA was separated into nt- and p-RNA using streptavidin coated magnetic beads. All three fractions were used for microarray analysis. For actinomycin-D experiments only tc-RNA was used prepared from cell before and 1 an 2h after addition of act-D. We used microarrays to analyze the effects of 1 and 3h of IFNalpha and gamma treatment in total cellular RNA Experiment Overall Design: NIH-3T3 cells ( 5th to 15th passage after thawing) were split from confluent plates 24h before start of the experiment. At the begin of the experiment about 80% confluency was reached. The experiment was started by applying fresh, prewarmed, CO2-equilibrated medium containing either 200ÂµM 4sU (for 1h labeling), 500ÂµM 4sU (for <=30min labeling) or 5Âµg/ml actinomycin-D.

2009-10-20 | E-GEOD-10011 | biostudies-arrayexpress

newly transcribed RNA (nt-RNA) for IFN alpha and gamma time course

2009-09-30 | GSE9975 | GEO

Expression data from NIH-3T3 cells used for half-life determination

Project description:Data from tc-, nt- and p-RNA as well as 1 and 2h of actinomycin-D treatment (5µg/ml) of NIH-3T3 cells used to determine half-lives. RNA was labeled for 15, 30 or 60 minutes with 4-thiouridine. After preparation of tc-RNA, thiol-labeled RNA was biotinylated using biot-HPDP and subsequently tc-RNA was separated into nt- and p-RNA using streptavidin coated magnetic beads. All three fractions were used for microarray analysis. For actinomycin-D experiments only tc-RNA was used prepared from cell before and 1 an 2h after addition of act-D. We used microarrays to analyze the effects of 1 and 3h of IFNalpha and gamma treatment in total cellular RNA Keywords: determination of RNA half-lives in NIH-3T3

2009-09-30 | GSE10011 | 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

Transcription profiling of mouse NIH-3T3 cells treated with interferon alpha or gamma

Project description:Expression data from NIH-3T3 cells treated with mock, 100 U/ml IFN alpha or 100 U/ml gamma for 1 or 3h on nt-RNA labeled for 30-60 min at different times of interferon treatment; Differential gene expression caused by IFN alpha or gamma was analyzed in newly transcribed RNA (nt-RNA) of NIH-3T3 cells treated for 1 or 3 h. RNA was labeled for 30 to 60 min and separated from total cellular RNA (tc-RNA) following Trizol RNA preparation and thiol-specific biotinylation; We used microarrays to analyze the effects of IFNalpha and gamma treatment in newly transcribed RNA (nt-RNA) Experiment Overall Design: NIH-3T3 cells (5th to 15th passage after thawing) were split 24 h before start of the experiment. When starting the experiment 80% confluency was reached. The experiment was started by applying fresh, prewarmed, CO2-equilibrated medium containing either mock, 100U/ml IFN alpha or 100 U/ml IFN gamma. Labeling was either started simultaneously, 30 or 150 minutes later.

2009-10-11 | E-GEOD-9975 | biostudies-arrayexpress

Identification of KSHV, EBV and cellular miRNA targets in human B-cells using RIP-Chip

Project description:RIP-Chip was performed on DG75-eGFP, DG75-10/12, BCBL-1, BL41, BL41 B95.8 and Jijoye using anti-human Ago2 (11A9) antibodies. Anti-BrdU antibodies were used as controls for DG75-eGFP, DG75-10/12 and BCBL-1. Total RNA was used as control for BL41, BL41 B95.8 and Jijoye. Samples were analyzed on Affymetrix Gene ST 1.0 Arrays (2 independent biological replicates / sample)

2011-02-04 | GSE17180 | GEO

Small RNA-Seq in ALKBH3 stable over expression and control HeLa cells

Project description:Total RNA (100 μg) were denatured at 70 °C for 5 min and separated by a 15% TBE-Urea gel with 20/100 or 10/60 oligo length standard ladder (Integrated DNA Technologies, Coralville, IA). After visualized by SYBR Gold (Ref. S11494 from Life Technologies), tRFs (15~50 nt) in gels were cut according to sizes and recovered by small RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA). The recovered tRFs was used to establish the library by NEB small RNA library preparation kit (E7330S).

2021-04-09 | GSE112870 | GEO

Small RNA seq in ALKBH3 stable over expresion and control HeLa cells

2021-04-21 | GSE113490 | GEO

CRISPR-Cas9 mediated editing of the CD46 gene in primary human B cells prior to their infection with Epstein-Barr virus (EBV) – analysis of CD46 transcripts after 24 hours

Project description:Adenoid B cells from anonymous human donors were isolated and nucleofected with RNP complexes targeting two loci within exon 2 of the CD46 gene, which encodes a cell surface protein. One hour after nucleofection, the same number of control cells (w/o-RNP) and CD46-RNP complex treated cells were infected with Epstein-Barr virus (strain 6008_B95-8) with a multiplicity of infection of 0.1. Newly transcribed RNAs were metabolically labeled with 4-thiouridine (4sU) for 60 min 23 hours after EBV infection. Twenty-four hours after the start of the experiment, 4sU-biotin-labeled, newly transcribed RNAs were thiol-specifically biotinylated and separated from unlabeled RNA using Streptavidin beads. RNA-seq experiments with the enriched RNA samples were performed to analyze the edited CD46 locus encompassing the RNP complex target sites. The experiment revealed that 24 hours after CRISPR-Cas9 mediated gene editing a substantial fraction of CD46 transcript contained indels and a larger deletion in between the two targets sites within exon 2 of CD46.

2020-10-18 | E-MTAB-9531 | biostudies-arrayexpress

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