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Series GSE60358 Query DataSets for GSE60358
Status Public on Apr 23, 2015
Title Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged.
Overall design To monitor nascent RNA within the mammalian Pol II complex, and its association with different CTD phosphorylation states, we employed mNET-seq methodology on HeLa cells, complemented with direct sequencing of chromatin-bound RNA (ChrRNA-seq). mNET-seq was preformed using the antibodies 8WG16, CMA602, CMA603 and CMA601, which are specific for unphosphorylated CTD, Ser2 phosphorylation, Ser5 phosphorylation and all CTD isoforms, respectively. In another experiment, to evaluate the effect of transcription termination factors in nascent RNA production by Pol II, mNET-seq and complemented with ChrRNA-seq was preformed on HeLa cells transfected with siRNA against PTBP1, CPSF73, CstF64+CstF64tau or Xrn2, and the gene profiles were compared with profiles from HeLa transfected with siRNA for Luciferase generated by the same protocol.
Contributor(s) Nojima T, Gomes T, Grosso AF, Kimura H, Dye MJ, Dhir S, Fonseca MC, Proudfoot NJ
Citation(s) 25910207, 37783853
Submission date Aug 12, 2014
Last update date Oct 31, 2023
Contact name Tomás Gomes
Organization name Instituto de Medicina Molecular
Street address Av. Professor Egas Moniz
City Lisboa
ZIP/Postal code 1649-028 Lisboa
Country Portugal
Platforms (2)
GPL11154 Illumina HiSeq 2000 (Homo sapiens)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
Samples (28)
GSM1474225 mNET_8WG16_rep1
GSM1474226 mNET_8WG16_rep2
GSM1474227 mNET_CMA602_rep1
BioProject PRJNA258089
SRA SRP045447

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Supplementary file Size Download File type/resource
GSE60358_RAW.tar 4.7 Gb (http)(custom) TAR (of BW)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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