Comment[ArrayExpressAccession] E-GEOD-43440 MAGE-TAB Version 1.1 Public Release Date 2013-01-29 Investigation Title Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response Comment[Submitted Name] Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response Experiment Description Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, miRNAs, and RNA-binding proteins. Here, we present Bru-Seq and BruChase-Seq to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory TNF. The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steadystate total RNA and should be useful in many biological settings. Analysis of the effect of TNF exposure in nascent gene expression and in transcript stability Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Ljungman Paulsen Veloso Prasad Bedi Ljungman Tsan Chang Tarrier Washburn Lyons Robinson Kumar-Sinha Wilson Ljungman Person First Name Mats Michelle Artur Jayendra Karan Emily Ya-Chun Ching-Wei Brendan Joseph Robert Daniel Chandan Thomas Mats Person Mid Initials T A G R E Person Email tenbroek@med.umich.edu Person Affiliation University of Michigan Person Address University of Michigan, NCRC, B520 Room 1346 2800 Plymouth Rd., Ann Arbor, Michigan, USA Person Roles submitter Protocol Name P-GSE43440-4 P-GSE43440-1 P-GSE43440-3 P-GSE43440-2 Protocol Description Library strategy: Bru-Seq Illumina Casava v1.8.2 software used for basecalling. Reads were mapped to the Human ribosomal DNA complete repeating unit (U13369.1) using bowtie v.0.12.8 and parameters -n 3 -k 1 -m 1 Reads that did not map to the ribosomal DNA were mapped to the hg19 genome using TopHat v1.4.1 and parameters M-bM-^@M-^Smin-isoform-fraction 0, --max-multihits 1, --no-closure-search, --no-coverage-search, --bowtie-n, --initial-read-mismatches 3 A coverage determination was then made for every base in the genome using Bedtools v.2.16.2 such that a base covered by one read was recorded as having a coverage of 1/read_length Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library. Genome_build: hg19 Supplementary_files_format_and_content: The columns of the abundance measurement files indicate: (1) chromosome; (2) start; (3) end; (4) name; (5) score; (6) strand; (7) gene length; (8) feature type M-bM-^@M-^S exon, intron, etc; (9) number of introns and exons; (10) feautre length; (11) hits count; (12) density M-bM-^@M-^S count/length; (13) RPKM - count/(length/1E3)/(mapped read count/1E6) Bromouridine (Aldrich) was added to the media to a final concentration of 2 mM and cells were incubated at 37M-0C for 30 min. Cells were then washed 3 times in PBS and either collected directly (nascent RNA, Bru-Seq) or chased in conditioned media containing 20 mM uridine for 6 hours at 37M-0C (6-hour old RNA, BruChase-Seq). For TNF treatments, recombinant human TNF-alpha (R&D Systems, Minneapolis, MN) was used in a concentration of 10 ng/ml from a 10 M-NM-