title: A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome.
abstract: BACKGROUND
  The initiating nucleotide found at the 5' end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. The currently available methods do not use targeted enrichment for the 5'end of primary transcripts, but rather attempt to deplete non-targeted RNA.
  
  
  RESULTS
  We developed a method, Cappable-seq, for directly enriching for the 5' end of primary transcripts and enabling determination of transcription start sites at single base resolution. This is achieved by enzymatically modifying the 5' triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli, achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and identified TSS in a microbiome.
  
  
  CONCLUSIONS
  Cappable-seq allows for the first time the capture of the 5' end of primary transcripts. This enables a unique robust TSS determination in bacteria and microbiomes.  In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.
authors.affiliation: New England Biolabs, 240 County Rd, Ipswich, MA, 01938, USA. ettwiller@neb.com.
authors.forename: Laurence
authors.initials: L
authors.lastname: Ettwiller
authors.affiliation: New England Biolabs, 240 County Rd, Ipswich, MA, 01938, USA. buswell@neb.com.
authors.forename: John
authors.initials: J
authors.lastname: Buswell
authors.affiliation: New England Biolabs, 240 County Rd, Ipswich, MA, 01938, USA. yigit@neb.com.
authors.forename: Erbay
authors.initials: E
authors.lastname: Yigit
authors.affiliation: New England Biolabs, 240 County Rd, Ipswich, MA, 01938, USA. schildkraut@neb.com.
authors.forename: Ira
authors.initials: I
authors.lastname: Schildkraut
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D000069196:Gastrointestinal Microbiome / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*; D024363:Transcription Initiation Site; D059467:Transcriptome
chemical_list: D012329:RNA, Bacterial