title: Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA.
abstract: Gene expression studies commonly examine total cellular RNA, which only provides information about its steady-state pool of RNA. It remains unclear whether differences in the steady-state reflects variable rates of transcription or RNA degradation. To specifically monitor RNA synthesis and degradation genome-wide, we developed Bru-Seq and BruChase-Seq. These assays are based on metabolic pulse-chase labeling of RNA using bromouridine (Bru). In Bru-Seq, recently labeled RNAs are sequenced to reveal spans of nascent transcription in the genome. In BruChase-Seq, cells are chased in uridine for different periods of time following Bru-labeling, allowing for the isolation of RNA populations of specific ages. Here we describe these methodologies in detail and highlight their usefulness in assessing RNA synthesis and stability as well as splicing kinetics with examples of specific genes from different human cell lines.
authors: Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA.
authors: Michelle T
authors: MT
authors: Paulsen
authors: Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA; Bioinformatics Program, University of Michigan, Ann Arbor, MI, USA.
authors: Artur
authors: A
authors: Veloso
authors: Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA.
authors: Jayendra
authors: J
authors: Prasad
authors: Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA; Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI, USA.
authors: Karan
authors: K
authors: Bedi
authors: Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA.
authors: Emily A
authors: EA
authors: Ljungman
authors: Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA.
authors: Brian
authors: B
authors: Magnuson
authors: Department of Pathology, University of Michigan, Ann Arbor, MI, USA; Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA.
authors: Thomas E
authors: TE
authors: Wilson
authors: Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA; Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI, USA. Electronic address: ljungman@umich.edu.
authors: Mats
authors: M
authors: Ljungman
mesh_terms: D000818:Animals; D001976:Bromouracil / Q000031:analogs & derivatives; D018389:Codon, Nonsense; D018076:DNA, Complementary / Q000235:genetics; D016368:Frameshift Mutation; D015894:Genome, Human; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D020014:K562 Cells; D007700:Kinetics; D058977:Molecular Sequence Annotation; D012326:RNA Splicing; D020871:RNA Stability; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA; D013194:Staining and Labeling; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: C497483:CD44 protein, human; D018389:Codon, Nonsense; D018076:DNA, Complementary; D018960:Hyaluronan Receptors; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D001976:Bromouracil; C006824:5-bromouridine; D014529:Uridine
keywords: RNA splicing; RNA stability; Transcription
references: DNA Res. 2008 Aug;15(4):241-51
references: 18611946
references: Genome Biol. 2009;10(3):R25
references: 19261174
references: Br J Cancer. 2006 Jan 30;94(2):318-22
references: 16421597
references: Cancer Res. 1989 Aug 1;49(15):4305-10
references: 2545339
references: Nat Struct Mol Biol. 2009 Feb;16(2):107-13
references: 19190664
references: Bioinformatics. 2012 Mar 15;28(6):884-5
references: 22285829
references: Genome Biol. 2010;11(10):R106
references: 20979621
references: Proc Natl Acad Sci U S A. 1984 Nov;81(22):7046-50
references: 6594679
references: Cancer Res. 1999 Jan 1;59(1):141-50
references: 9892199
references: Br J Haematol. 2006 Apr;133(1):98-102
references: 16512835
references: Nucleic Acids Res. 1993 Mar 11;21(5):1225-9
references: 8464707
references: Bioinformatics. 2010 Mar 15;26(6):841-2
references: 20110278
references: Genome Res. 2012 May;22(5):947-56
references: 22369889
references: Mol Cell. 2011 Sep 16;43(6):904-14
references: 21925379
references: Proc Natl Acad Sci U S A. 2013 Feb 5;110(6):2240-5
references: 23345452
references: Exp Cell Res. 1997 Aug 1;234(2):498-506
references: 9260920
references: Genes Dev. 2011 Dec 1;25(23):2502-12
references: 22156210
references: Mol Cell. 2013 Apr 25;50(2):212-22
references: 23523369
references: Science. 2008 Dec 19;322(5909):1845-8
references: 19056941
references: Genome Res. 2012 Jul;22(7):1350-9
references: 22466169
references: Bioinformatics. 2009 May 1;25(9):1105-11
references: 19289445
references: J Biomed Biotechnol. 2009;2009:860761
references: 20145706
references: Cell Rep. 2012 Oct 25;2(4):1025-35
references: 23062713
references: Nature. 2011 Jan 20;469(7330):368-73
references: 21248844
references: Nature. 2011 May 19;473(7347):337-42
references: 21593866
references: Bioinformatics. 2009 Aug 15;25(16):2078-9
references: 19505943
references: Nat Biotechnol. 2011 May;29(5):436-42
references: 21516085
references: Cell. 2009 Feb 20;136(4):763-76
references: 19239894