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| Status |
Public on Jan 06, 2012 |
| Title |
H3K9me3 nucleosome-IP from eri-1(mg366) mutant C. elegans that underwent ego-1 RNAi |
| Sample type |
SRA |
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| Source name |
adult C. elegans
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| Organism |
Caenorhabditis elegans |
| Characteristics |
chip antibody: anti-H3K9me3 antibody
chip antibody manufacturer: Abcam
chip antibody catalog #: ab8898
genetic background: N2
strain: eri-1(mg366)
developmental stage: adult
barcode (first 3 nt for fastq files of chip-seq libraries, first 4 nt for fastq files of small rna libraries): GCT
RNAi target: chrI: 7655024-7654481 (ego-1)
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| Treatment protocol |
50-100 μl frozen embryo pellets or 100 - 200 μl frozen adult worm pellets were used for each nucleosome immunoprecipitation experiment. Crushed pellets (pulverized by grinding in liquid nitrogen with a mortar and pestle) were resuspended in 1 ml 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, 1x HALT protease and phosphatase inhibitor cocktail (Thermo Scientific)). To crosslink, formaldehyde was added to the resulting crude extract to a final concentration of 2%, followed by incubation at room temperature for 15 min. 0.1 ml 1 M Tris.Cl (pH 7.5) was then added to quench formaldehyde. Lysate was spun (15,000xg for 1 min). Pellets were washed with ice-cold buffer A and then resuspended in 0.3 ml buffer A with 2 mM CaCl2. Micrococcal nuclease (Roche) was added to the lysate to a final concentration of 0.3 U/μl, followed by 5 minutes incubation at 37°C (inverting the tube several times per minute). Micrococcal Nuclease (MNase) digestion was optimized so that approximately 70% of DNA entered the mono-nucleosome band, with the remainder predominantly in di- and trinucleosome bands. Digestion reactions were stopped by adding EGTA to a final concentration of 20 mM. After centrifugation (15,000xg, 1 min) pellets were washed with ice-cold RIPA buffer (1xPBS, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS, 1x HALT protease and phophatase inhibitor and 2 mM EGTA), resuspended in 0.8 ml ice-cold RIPA buffer, and solubilized by sonication (Microson TM XL 2000 sonicating machine with a micro tip, output level: 5, three times of 30-second sonication). Sonication was used here to efficiently solubilize crosslinked chromatin without further fragmenting the chromatin (data not shown). Crude lysate was cleared by centrifugation at 15,000xg for 2 min. 80 μl of the supernatant was used to make "IP input" nucleosome libraries. The remaining supernatant was used for IP, adding 2 μg of anti-H3K9me3 antibody (Abcam, ab8898) and agitating gently at 4°C overnight before 50 μl of protein A Dynabeads (10% slurry in 1x PBS buffer) was added and the resulting mixture shaken for 2hr at 4°C . The beads were then washed four times (four minutes each) with ice-cold 600 μl LiCl washing buffer (100mM Tris.Cl, pH 7.5, 500mM LiCl, 1%NP-40, 1% Sodium deoxycholate), transferring to a new tube after the first wash. To elute the immunoprecipitated nucleosome and reverse crosslinks, beads were incubated with 450 μl worm lysis buffer (0.1M Tris.Cl, pH 8.5, 100 mM NaCl, 1% SDS) containing 200 μg/ml protease K at 65°C for four hours with agitation every 30 minutes (total nucleosome aliquots were treated similarly to reverse crosslink), followed by organic extraction and DNA precipitation.
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| Growth protocol |
RNAi by feeding was carried out as previously described (Timmons, L. & Fire, A. Specific interference by ingested dsRNA. Nature 395, 854 (1998).)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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 (100 μM each, Roche), and 1x NEB buffer 1 at 12°C for 15 min. Nucleosome core DNA was then ligated to previously annealed DNA oligos 5’ Pi-/barcode/-AGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-OH 3’ and 5’ OH-CCCTACACGACGCTCTTCCGATCT-/barcode/-OH 3’) (NEB Quick Ligation Kit). Nucleosome core DNA captured by linker oligos 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 were 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. 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) and used for Illumina GAIIx.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
SG0910_4lib.bed; genome build: C. elegans, May 2008 (WS190/ce6)
After removing the 3-nt barcode, the first 25-nt sequence for each tag was used to align to the C. elegans genome (WS190 version) using Bowtie 0.12.7. Only perfect alignments were included in the bed file.
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| Submission date |
Dec 29, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Sam Guoping Gu |
| E-mail(s) |
[email protected]
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| Organization name |
Rutgers University
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| Department |
Molecular Biology and Biochemistry
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| Lab |
Sam Gu
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| Street address |
Nelson Labs A125, 604 Allison Road
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| City |
Piscataway |
| State/province |
NJ |
| ZIP/Postal code |
08854 |
| Country |
USA |
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| Platform ID |
GPL13776 |
| Series (1) |
| GSE32631 |
Amplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint |
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| Relations |
| SRA |
SRX113600 |
| BioSample |
SAMN00769190 |
| Named Annotation |
GSM855078_SG0910_4lib.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM855078_SG0910_4lib.bed.gz |
80.8 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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