 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 31, 2009 |
| Title |
H3K4me2/3 N2 chipSeq |
| Sample type |
SRA |
| |
|
| Source name |
synchronized young adult population of wild type strain N2
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
developmental stage: young adult
chip antibody: anti-H3K4me2/3
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nucleosome core preparation Synchronized populations of C. elegans (wild type strain N2 or mutant strain zim-2 (tm574)) were fed on E.coli OP50 (Brenner, 1974). Young adult worms were flash frozen and ground to fine powder in liquid nitrogen. Worm grinds were directly resuspended in buffer A (15 mM Hepes-Na, pH 7.5, 60 mM KCl, 15 mM NaCl, 0.15 mM beta-mecaptoethonal, 0.15 mM spermine, 0.15 mM spermidine, 0.34 M sucrose), 0.5 mM PMSF, 1/100 dilution of protease set III (Cal Biochem), 1 mM DTT, and 2 mM CaCl2. Micrococcal nuclease (MNase) (Roche), resuspended as 2 mg/ml in 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, and 1 mM DTT, was added to the C. elegans extract. Titrations of enzyme concentration at a series of time points were performed to find a condition where the majority of chromatin was digested to mono- and di-nucleosomes. Digestions (at 37°C) were stopped by adding EGTA to a final concentration of 20 mM. Nucleosomes were solubilized by slowly adding drops of 2 M NaCl to a final concentration of 450 mM. The extract from N2 animals was further fractionated in a 5-30% sucrose gradient (Beckman SW-41 rotor, 28,500 rpm for 18 hours). Mononucleosome fractions were determined by DNA extraction and gel analysis, dialyzed in PBS buffer (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, and pH 7.4) containing 1 mM PMSF, and stored at -80°C. The ability of zim-2(tm574) mutant animals to enrich H3K9me3 on the pairing center arm of chromosome V was confirmed using a simplified protocol omitting the gradient fractionation. Nucleosome immunoprecipitation For each immunoprecipitation reaction, 2.5 μg antibody to either histone H3 (di- and trimethylated lysine 4, Abcam 6000) or histone H3 (trimethylated lysine 9, Abcam 8898) was captured to 10 μl Dynabead® conjugated with protein A (Invitrogen) that had been washed and resuspended in 95 μl PBS buffer with 0.01% Tween-20. Nucleosome core fraction (N2) or crude MNase-treated extract (zim-2) was mixed with the conjugated antibodies with constant rotation at 4 °C for 6 hours. The immunoprecipitant was washed thrice with 1 ml IP washing buffer (100 mM Hepes-Na, pH 7.5, 450 mM NaCl, 1% NP-40) at 4 °C. The washing mix was rotated for three minutes for each wash. Immunoprecipitated nucleosome core DNA was isolated by incubating the beads with 400 μl lysis buffer (100 mM Tris-Cl, pH 7.5, 0.1 M NaCl, 1% SDS, 50 mM EDTA, 0.2 mg/ml protease K) at 65 °C for 1 hour, followed by organic extraction and DNA precipitation. DNA library preparation Nucleosome core DNA was treated with 0.5 U/μl T4 polynucleotide kinase (NEB) in 1x T4 DNA ligase buffer (NEB, containing 1 mM ATP) at 37 °C for 1 hour to phosphorylate 5’ ends and dephosphorylate 3’ ends. Ends were then blunted in a reaction containing 0.75 U/μl of T4 DNA polymerase (NEB), dNTP (2mM each, Roche), and 1x NEB buffer 1 at 12 °C for 15 min. Nucleosome core DNA was then ligated to previously annealed DNA oligos AF-SG-133 (5’ Pi-AGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-OH 3’) and AF-SG-134 (5’ OH-CCCTACACGACGCTCTTCCGATCT-OH 3’) (NEB Quick Ligation Kit). Nucleosome core DNA captured by linker oligos <search for "oligoes"> was purified using 6 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea, followed by PCR amplification with primers AF-SG-135 (5’ OH-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-OH 3’) and AF-SG-137 (5’ OH-CAAGCAGAAGACGGCATACGAGCT-OH 3’). A series of PCR reactions with increasing cycle numbers was then carried out; we carefully choose cycle numbers for which product levels have not saturated (i.e. product levels still able to increase substantially with additional cycles); this ensures that the majority of amplified segments are still annealed to a true complement and avoids reannealing-distortion in the resulting sequence libraries (Parameswaran et al., 2007). . After separating PCR products on 2 % agarose gels, DNA bands of the expected size (~210-240 bp) were extracted (QIAchange® Gel Extraction Kit (Invitrogen); omitting the 50°C heating step), followed by massive parallel DNA sequencing (Illumina GAII).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
nucleosome core DNA
anit-H3K4me2/3 precipitated nucleosome core DNA
alignment in UCSC genome browser bed file format
library strategy: Micrococcal nuclease digestion and chromatin immunoprecipitation were used to obtain distinct populations of single nucleosome cores
library selection: Mnase and ChIP
|
| Data processing |
alignment:The first 25 nt of each raw sequencing read was used to map the nucleosome end to the C. elegans genome (WS190) using Eland (Illumina) (Bentley et al., 2008) To avoid ambiguity, only perfect and unique matches were used. For each unambiguous read, a putative nucleosome core sequence was defined by extending the sequence read to 147 bp.
processed: For each unambiguous read, a putative nucleosome core sequence was defined by extending the sequence read to 147 bp.
|
| |
|
| Submission date |
Jul 23, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Sam Guoping Gu |
| E-mail(s) |
ggu@dls.rutgers.edu
|
| Organization name |
Rutgers University
|
| Department |
Molecular Biology and Biochemistry
|
| Lab |
Sam Gu
|
| Street address |
Nelson Labs A125, 604 Allison Road
|
| City |
Piscataway |
| State/province |
NJ |
| ZIP/Postal code |
08854 |
| Country |
USA |
| |
|
| Platform ID |
GPL9269 |
| Series (1) |
| GSE17284 |
Partitioning the C. elegans genome by nucleosome modification, occupancy, and positioning |
|
| Relations |
| SRA |
SRX012297 |
| BioSample |
SAMN00004581 |
| Named Annotation |
GSM432721_H3K4me2:3_N2_chipSeq.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM432721_H3K4me2:3_N2_chipSeq.bed.gz |
9.5 Mb |
(ftp)(http) |
BED |
| GSM432721_H3K4me2:3_N2_chipSeq.txt.gz |
59.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
|
|
|
|
 |
