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| Status |
Public on Aug 30, 2006 |
| Title |
Genomewide demarcation of RNA PolII transcription units by physical fractionation of chromatin- ORF-INT enrichment |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
|
| Summary |
Epigenetic modifications of chromatin serve an important role in regulating the expression and accessibility of genomic DNA. We report here a genomewide approach for fractionating yeast chromatin into two functionally distinct parts, one containing RNA polymerase II transcribed sequences, and the other comprising noncoding sequences and genes transcribed by RNA polymerases I and III. Noncoding regions could be further fractionated into promoters and segments lacking promoters. The observed separations were apparently based on differential crosslinking efficiency of chromatin in different genomic regions. The results reveal a genomewide molecular mechanism for marking promoters and genomic regions that have a license to be transcribed by RNA polymerase II, a previously unrecognized level of genomic complexity that may exist in all eukaryotes. Our approach has broad potential use as a tool for genome annotation and for the characterization of global changes in chromatin structure that accompany different genetic, environmental, and disease states.
Keywords: Genomewide mapping of regulatory elements through differential fractionation of crosslinked chromatin based on nucleosome occupancy.
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| Overall design |
ORF-INT enrichment- Whole cells were fixed by addition of 37% formaldehyde/11% methanol to the growth medium to a final concentration of 1% formaldehyde at 30°C for 30 min. Glycine was added to 125mM from a 2.5 M stock and incubated for 5 min. The cells were centrifuged in a Sorvall RT7 at 3,000 rpm for 5 min at 4°C and
washed twice with PBS and once with sterile water. Without
reversing crosslinks, extracts were prepared by glass-bead disruption,
sonication (fragment size 200-2,000 bp, peak at 900
bp), and standard phenol-chloroform extraction.
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| Web link |
https://genome.unc.edu/cgi-bin/SMD/publication/viewPublication.pl?pub_no=2&16803
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| Citation(s) |
12750471 |
| |
| Submission date |
Aug 28, 2006 |
| Last update date |
Jan 18, 2013 |
| Contact name |
Jason D Lieb |
| E-mail(s) |
jlieb@bio.unc.edu
|
| Phone |
919-843-3229
|
| URL |
http://www.bio.unc.edu/faculty/lieb/labpages/default.shtml
|
| Organization name |
University of North Carolina at Chapel Hill
|
| Department |
Biology
|
| Lab |
Jason Lieb
|
| Street address |
203 Fordham Hall
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
| |
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| Platforms (3) |
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| Samples (5)
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| GSM132369 |
Exp. #23, ORF+Int enr. Fixed Nuclear Extract (1% 30 min) *IP #1* REVERSED BEFORE P/C vs Fixed Nuclea |
| GSM132370 |
Exp. #24, ORF+Int enr. Fixed Nuclear Extract (1% 30 min) *IP #2* REVERSED BEFORE P/C vs Fixed Nuclea |
| GSM132371 |
Exp. #25, ORF+Int enr. Channel Swap for jdl_g_111E |
| GSM132372 |
Exp. #26, ORF+Int enr. Fixed Nuclear Extract (1% 30 min) *IP #3* REVERSED BEFORE P/C vs Fixed Nuclea |
| GSM132373 |
Exp. #27, ORF+Int enr. Fixed Nuclear Extract (1% 30 min) *Mock IP, No Ab* REVERSED BEFORE P/C vs Fix |
|
| Relations |
| BioProject |
PRJNA96853 |
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