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| Status |
Public on Aug 30, 2006 |
| Title |
Genomewide demarcation of RNA PolII transcription units by physical fractionation of chromatin- ALL Arrays |
| Organism(s) |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| 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 demarcation of RNA PolII transcription units by physical fractionation of chromatin- Intergenic enrichment
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| Overall design |
This series contains all the arrays associated with Nagy et al (PNAS 2003 May 27;100(11):6364-9). The arrays can be divided into four logical sets: Intergenic enrichment (GSE5648), ORF enrichment (GSE5649), ORF-Intergenic enrichment (GSE5650), and Neutral enrichment (GSE5651). Shown with each logical set is a series accession number containing the arrays associated with that set. Below is an explanation for the experimental procedure used to derived the samples for this manuscript.
Standard DNA Preparation- (Experiments 1–8) All DNA was prepared by glass-bead disruption of cells and a standard phenol-chloroform extraction. The extract was centrifuged for 5 s at 14,000 x g, and the supernatant was sonicated and subsequently phenol-chloroform extracted.
Intergenic Enrichment Procedure- (Experiments 9–27) 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 125 mM 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.
ORF Enrichment Procedure- (Experiments 31–32) Cells were crosslinked as above, and isolated nuclei were used to prepare solubilized chromatin. Crosslinks were then reversed by incubation at 65°C, and DNA was prepared. In initial experiments (nos. 28–30), immunoprecipitation using antimethyl-lysine histone H3 antibody was performed before the crosslinks were reversed. However, the IPs were not required for ORF enrichment .
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| Web Link |
https://genome.unc.edu/cgi-bin/SMD/publication/viewPublication.pl?pub_no=2&13462
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| Contributor(s) |
Nagy PL, Cleary ML, Brown PO, Lieb JD |
| Citation(s) |
12750471
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| Submission date |
Aug 29, 2006 |
| Contact name |
Jason D Lieb |
| E-mail(s) |
jlieb@bio.unc.edu
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| Phone |
919-843-3229
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| URL |
http://www.bio.unc.edu/faculty/lieb/labpages/default.shtml
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| Organization name |
University of North Carolina at Chapel Hill
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| Department |
Biology
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| Lab |
Jason Lieb
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| Street address |
203 Fordham Hall
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
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| Platforms (3) |
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| Samples (32)
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| GSM132347 |
Exp. #10, Intergenic enrichment. Fixed Whole Extract (1% 15 min) vs Whole Extract Nagy 4-25-02 |
| GSM132348 |
Exp. #14, Intergenic enrichment. Fixed Whole Extract (1% 60 min) vs Whole Extract Nagy 4-25-02 |
| GSM132349 |
Exp. #17, Intergenic enrichment. Fixed Whole Extract (5% 15 min) vs Whole Extract. Nagy 4-25-02 |
| GSM132350 |
Exp. #28, ORF enr. Fixed Nuclear Extract (1% 30 min) *IP #1* REVERSED BEFORE P/C vs S288C Genomic |
| GSM132351 |
Exp. #02, Neutral. Whole Extract vs Whole Extract Nagy 2-05-02 |
| GSM132352 |
Exp. #03, Neutral. Whole Extract CSH Prep vs S288C Genomic DNA |
| GSM132353 |
Exp. #04, Neutral. Whole Extract vs 50 ng S288C Genomic DNA JDL 2-28-02 |
| GSM132354 |
Exp. #05, Neutral. 0.1 ng S288C Genomic DNA, amplified vs 50ng of same DNA, amplified |
| GSM132355 |
Exp. #06, Neutral. DNA prepared from Nuclear Extract vs Whole Extract, Nagy 2-05-02 |
| GSM132356 |
Exp. #07, Neutral. Whole Extract FIXED AFTER P/C Prep vs Whole Extract, Nagy 2-05-02 |
| GSM132357 |
Exp. #08, Neutral. Fixed Whole Extract, Reversed before P/C vs Whole Extract, Nagy 2-05-02 |
| GSM132358 |
Exp. #09, Intergenic enrichment. Fixed Whole Extract (1% 15 min, High Phenol/Aq) vs Whole Extract |
| GSM132359 |
Exp. #11, Intergenic enrichment. Fixed Whole Extract (1% 30 min) vs Whole Extract JDL 2-28-02 |
| GSM132360 |
Exp. #12, Intergenic enrichment. Fixed Whole Extract (1% 15 min) vs Whole Extract Nagy 2-05-02 |
| GSM132361 |
Exp. #13, Intergenic enrichment. Fixed Whole Extract (1% 30 min) REVERSED AFTER P/C prep vs ... |
| GSM132362 |
Exp. #15, Intergenic enrichment. Fixed Whole Extract (1% 60 min, break, 30 mins @ 37, then P/C) ... |
| GSM132363 |
Exp. #16, Intergenic enrichment. Fixed Whole Extract (1% 60 min, 30 mins @ 37 w/ProtK, P/C) vs ... |
| GSM132364 |
Exp. #18, Intergenic enrichment. Fixed Spheroplasts Extract vs Whole Extract. Nagy 2-05 |
| GSM132365 |
Exp. #19, Intergenic enrichment. Fixed Spheroplasts Extract (Incubted in NIB on ice o/n) vs ... |
| GSM132366 |
Exp. #20, Intergenic enrichment. Fixed Nuclear Extract (1% 30 min), used as ref in jdl_g_111E vs ... |
| GSM132367 |
Exp. #21, Intergenic enrichment. Fixed Nuclear Extract (1% 30 min), used as ref for jdl_g_099K vs... |
| GSM132368 |
Exp. #22, Intergenic enrichment. Fixed Nuclear Extract (1% 30 min, samples from jdl_g_50J and 52J... |
| 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 |
| GSM132374 |
Exp. #29, ORF enr. Fixed Nuclear Extract (1% 30 min) *IP #2* REVERSED BEFORE P/C vs S288C Genomic |
| GSM132375 |
Exp. #30, ORF enr. Fixed Nuclear Extract (1% 30 min) *IP #4* REVERSED BEFORE P/C vs S288C Genomic DN |
| GSM132376 |
Exp. #31, ORF enr. Fixed Nuclear Extract (1% 30 min, same as jdl_g_112J sample but REVERSED ... |
| GSM132377 |
Exp. #32, ORF enr. Fixed Nuclear Extract (1% 30 min, same as jdl_g_112J sample but REVERSED ... |
| GSM132381 |
Exp. #01, Neutral. S288C Genomic DNA, 03/20/00 vs S288C Genomic DNA 03/20/00 #2 |
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| Supplementary data files not provided |
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