|
| Status |
Public on Jan 01, 2010 |
| Title |
OGT S L1 RL2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ogt-1(ok430) mutant ChIP
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: ogt-1(ok430) mutant
developmental stage: L1 larvae
condition: starved
antibody: RL2: O-GlcNAc (ab2739, Abcam)
|
| Treatment protocol |
Starved L1 animals were wild type or one of two mutant strains: loss of the O-GlcNAc transferase ogt-1(ok430) and loss of the O-GlcNAcase oga-1(ok1207).
|
| Growth protocol |
Embryos were isolated from wild type and mutant gravid adults by bleach treatment and shaken gently with good aeration in sterile buffer to hatch and collected 48 hours later.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
C. elegans larvae were disrupted and fixed by brief sonication in 1% formaldehyde followed by incubation for 20 min. at room temperature. After washing, lysates were sonicated again to shear the DNA to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation.
Genomic DNA regions of interest were isolated using an antibody against one of the following epitopes: the unmodified and Ser-5-P-modified heptad repeat (YSPTSPS) and of the C-terminal domain (CTD) of RNA PolII (8WG16 MMS-126R, Covance); the Ser-2-phosphorylated and non-modified heptad repeat of the CTD domain (ab5095, Abcam); O-linked N-acetylglucosamine modified peptides (RL2; ab2739, Abcam), HGAC85-derived O-linked N-acetylglucosamine {Turner et al., 1990, #40278} (MA1-076, Thermo Scientific), and Ser/Thr-O-linked N-acetylglucosamine (CTD 110.6; MMS-248R, Covance).
|
| Label |
biotin
|
| Label protocol |
ChIP and input DNAs were amplified by whole-genome amplification (WGA) using the GenomePlex WGA Kit (Sigma). The resulting amplified DNAs were purified, quantified, and tested by qPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. Amplified DNAs were fragmented and labeled using the DNA Terminal Labeling Kit (Affymetrix).
|
| |
|
| Channel 2 |
| Source name |
Wild type input chromatin
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: wild type
developmental stage: L1 larvae
condition: starved
antibody: none
|
| Treatment protocol |
Starved L1 animals were wild type or one of two mutant strains: loss of the O-GlcNAc transferase ogt-1(ok430) and loss of the O-GlcNAcase oga-1(ok1207).
|
| Growth protocol |
Embryos were isolated from wild type and mutant gravid adults by bleach treatment and shaken gently with good aeration in sterile buffer to hatch and collected 48 hours later.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
C. elegans larvae were disrupted and fixed by brief sonication in 1% formaldehyde followed by incubation for 20 min. at room temperature. After washing, lysates were sonicated again to shear the DNA to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation.
Genomic DNA regions of interest were isolated using an antibody against one of the following epitopes: the unmodified and Ser-5-P-modified heptad repeat (YSPTSPS) and of the C-terminal domain (CTD) of RNA PolII (8WG16 MMS-126R, Covance); the Ser-2-phosphorylated and non-modified heptad repeat of the CTD domain (ab5095, Abcam); O-linked N-acetylglucosamine modified peptides (RL2; ab2739, Abcam), HGAC85-derived O-linked N-acetylglucosamine {Turner et al., 1990, #40278} (MA1-076, Thermo Scientific), and Ser/Thr-O-linked N-acetylglucosamine (CTD 110.6; MMS-248R, Covance).
|
| Label |
biotin
|
| Label protocol |
ChIP and input DNAs were amplified by whole-genome amplification (WGA) using the GenomePlex WGA Kit (Sigma). The resulting amplified DNAs were purified, quantified, and tested by qPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. Amplified DNAs were fragmented and labeled using the DNA Terminal Labeling Kit (Affymetrix).
|
| |
|
| |
| Hybridization protocol |
Labeled DNAs were hybridized to Affymetrix C. elegans GeneChip Tiling 1.0 arrays at 45o C overnight.
|
| Scan protocol |
Arrays were washed and scanned.
|
| Description |
OGT S L1 RL2 ChIP
N2 S L1 Input Chromatin
|
| Data processing |
The resulting CEL files were analyzed using Affymetrix TAS software.
Thresholds were selected, and the resulting BED files were analyzed using Genpathway proprietary software that provides comprehensive information on genomic annotation, peak metrics and sample comparisons for all peaks (intervals). To determine genes most strongly linked to O-GlcNAc associated (RL-2 reactive) chromatin regions, the input-normalized ChIP signal intensity for each probe from mutants unable to O-GlcNAcylate protein substrates (ogt-1(ok430)) was subtracted from that of mutants that are unable to remove GlcNAc from substrates (oga-1(ok1207)). A value at, or above, 2.2 was used to threshold resulting data in order to determine O-GlcNAc peaks. Bioinformatic analysis (GenPathway) was used to identify genes associated with RL2 peaks above threshold and located within 2 kb of the transcriptional start site (genome version, WS195). Application of these criteria resulted in the identification of 828 genes associated with O-GlcNAc-positive chromatin.
The .bar files are two-column text files that list the chromosomal location and signal intensity for each probe on the array. The .bed files are a three-column file that lists the chromosomal number, start, and stop points for all peaks above threshold.
|
| |
|
| Submission date |
Oct 16, 2009 |
| Last update date |
Oct 16, 2009 |
| Contact name |
Michael Krause |
| E-mail(s) |
mwkrause@helix.nih.gov
|
| Phone |
(301) 402-4633
|
| Organization name |
NIH
|
| Department |
LMB/NIDDK
|
| Lab |
Krause
|
| Street address |
5 Center Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL5634 |
| Series (2) |
| GSE18132 |
Dynamic O-GlcNAc cycling at promoters of C. elegans genes regulating Longevity, Stress, and Immunity |
| GSE18611 |
O-GlcNAc Chromatin Marks from C. elegans L1 animals |
|
