Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH
Abstract
Visualization of the transcriptome and the nuclear organization in situ has been challenging for single-cell analysis. Here, we demonstrate a multiplexed single-molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA seqFISH and immunofluorescence. This nascent transcriptome-profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts. The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories, and their organization in individual cells is highly variable. Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2 hr in mouse embryonic stem cells, as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured.
Additional Information
© 2018 Elsevier Inc. Received 27 June 2017, Revised 7 November 2017, Accepted 15 May 2018, Available online 7 June 2018Published: June 7, 2018. We thank A. Raj for discussion on the intron experiments; W. Reik, B. Simons, S. Rulands, and H. Lee for discussion on the 2-hr oscillation; M. Elowitz, M. Guttman, and M. Lawson for comments and suggestions on the manuscript; the Elowitz Lab and the Reik Lab for kindly providing cell lines; and the Gradinaru Lab for use of Imaris software. Y.T. is supported by a graduate fellowship from the Nakajima Foundation. This project is funded by NIHEB021240, HD075605, TR01 OD024686, and MH113508 and a Paul G. Allen Foundation Discovery Center grant. Data and Software Availability: MATLAB code for barcode calling and all image analysis steps: https://github.com/CaiGroup/Intron-SeqFish Raw data of TAS counts and mRNA counts used in the analysis presented in this article can be found in Table S1. Author Contributions: S.S., Y.T., W.Z., and L.C. designed the intron seqFISH experiments. W.Z., S.S., and E.L. performed the initial intron seqFISH experiments with the help of N.K., E.J.L., and M.A. Y.T. and C.-H.L.E. optimized and Y.T. collected the intron seqFISH data. S.S. and J.Y. carried out the pulse chase experiments. C.C. and C.K. built the automated fluidics. Y.T., S.S., and L.C. carried out the analysis. S.S., Y.T., W.Z., and L.C. wrote the manuscript. The authors declare no competing interests.Attached Files
Accepted Version - nihms970209.pdf
Submitted - 339234.full.pdf
Supplemental Material - mmc1.xlsx
Supplemental Material - mmc2.csv
Supplemental Material - mmc3.csv
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Additional details
- Alternative title
- Dynamics and spatial genomics of the nascent transcriptome in single mESCs by intron seqFISH
- PMCID
- PMC6046268
- Eprint ID
- 86889
- Resolver ID
- CaltechAUTHORS:20180607-131657607
- Nakajima Foundation
- NIH
- EB021240
- NIH
- HD075605
- NIH
- TR01 OD024686
- NIH
- MH113508
- Paul G. Allen Foundation
- Created
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2018-06-07Created from EPrint's datestamp field
- Updated
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2022-03-18Created from EPrint's last_modified field