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| Status |
Public on Jan 01, 2015 |
| Title |
H3K9me3_ChIP_WT |
| Sample type |
SRA |
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| Source name |
whole adult animals
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| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype: wild type (N2)
antibody: anti-Pol II H3K9me3 antibody (ab8898, Abcam)
developmental stage: young adult
barcode: AGC
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| Treatment protocol |
Fore all samples, young adult worms were flash frozen and ground to fine powder in liquid nitrogen using mortar and pestle.
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| Growth protocol |
Synchronized populations of C. elegans (wild type strain N2 or mutant strains ) were cultured on NGM plates at 19°C with E.coli OP50 as food (Brenner, 1974).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq experiments (sample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21): 100-200 μl frozen synchronized young adult worm pellets were used for each chromatin immnuoprecipitation experiment. Crushed pellets (pulverized by grinding in liquid nitrogen with a mortar and pestle) were resuspended in 1ml of pre-chilled RIPA buffer (1X PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1X HALT combined protease and phosphatase inhibitor cocktail [ThermoScientific]). To crosslink, formaldehyde was added to the crude extract to a final concentration of 2%. The lysate was rotated at 4°C for 10 minutes. 0.1 ml of 1M Tris-HCl (pH 7.5) was added to quench formaldehyde. Lysate was spun at 6000 x g for 30 second and the supernatant was removed. The pellet was resuspended in 0.5ml of pre-chilled RIPA buffer. The lysate was transferred to a 1.5 ml TPX tube (Diagenode) and sonicated (Bioruptor [Diagenode], output level: high, interval: 0.5, three times of 8-minute sonication). 50 μl of the lysate was used to make IP input library. For each immumoprecipiation experiment, 2 μg of anti-H3K9me3 (ab8898, Abcam), anti-Pol II-8WG16 (ab817, Abcam), anti-Pol II-S2 (ab5095, Abcam), or anti-Pol II-S5 (ab5131, Abcam) antibody was added to 400 μl of the lysate (containing approximately 50 μg DNA). The IP mix was rotated overnight at 4°C. 50 μl of Protein A Dynabeads (Life Technology) was added and rotated for another 2 hours. The beads were then washed three times with 800 μl ice-cold LiCl washing buffer (100 mM Tris-HCl, pH7.5, 500 mM LiCl, 1% NP-40 and 1% sodim deoxycholate). To elute the immunoprecipitation product and reverse crosslink, beads were incubated with 400 μl of worm lysis buffer (0.1 M Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, and 200 μg /ml of proteinase K) at 65 °C for 2 hours with agitation every 30 minutes (IP input lysate were treated similarly to reverse crosslink) and then subject to organic extraction and DNA precipitation.
small RNA-seq experiments (sample 18 and 19): Small RNA was extracted from frozen synchronized adult worm pellets (100-200 μl) using mirVana miRNA isolation kit (Life technologies).
pre-mRNA-seq experiments (sample 11 and 12): Worm grinds were resuspended in 1ml of pre-chilled RIPA buffer (1xPBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1X HALT combined protease and phosphatase inhibitor cocktail [ThermoScientific]). To cross-link, formaldehyde was added to the crude extract to a final concentration of 2%. The lysate was rotated at 4 °C for 10 minutes. 0.1 ml of 1M Tris-HCl (pH 7.5) was added to quench formaldehyde. Lysate were spun at 6000 x g for 30 second and the supernatant was removed. The pellet was resuspended in 0.5ml of pre-chilled RIPA buffer. The lysate was transferred to a 1.5 ml TPX tube (Diagenode) and sonicated (Bioruptor [Diagenode], output level: high, interval: 0.5, three times of 8-minute sonication). For each immumoprecipiation experiment, 2 μg anti-Pol II-S2 (ab5095, Abcam) antibody was added to 400 μl of the lysate (approximately 50 μg DNA). The IP mix was rotated overnight at 4 °C . 50 μl of Protein A Dynabeads (Life Technology) was added and rotated for another 2 hours. The beads were then washed three times with 800μl ice-cold LiCl washing buffer (100 mM Tris-HCl, pH7.5, 500 mM LiCl, 1% NP-40 and 1% sodim deoxycholate). To elute the immunoprecipitation product and reverse corss-links, beads were incubated with 400 μl of worm lysis buffer (0.1 M Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, and 200 μg /ml of proteinase K) at 65 °C for 2 hours with agitation every 30 minutes (IP input lysate were treated similarly to reverse cross-link samples) and then subject to phenol/Chloroform extraction, RNA/DNA precipitation, DNase I treatment, phenol/Chloroform extraction, and RNA precipitation. RNA was then fragmentated using Fragmentation Reagents (Ambion).
mRNA-seq experiments (sample 13, 14, 15, 16, and 17): Total RNA was extracted from frozen synchronized adult worm pellets (100-200 μl) using Trizol (Life Technologies). mRNA was selected from total RNA using Poly(A)Purist MAG kit (Life Technologies). mRNA was then fragmentated using Fragmentation Reagents (Ambion). 0.5-1 μg of mRNA was used for each mRNA-seq library.
ChIP-seq (sample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21): Same as described in Gu SG et. al. 2012 (PMID: 22231482).
pre-mRNA-seq (sample 11 and 12): The fragmented RNA was treated with T4 polynucleotide kinase (New England Biolabs). The 3’ end of RNA was ligated to an adenylated DNA oligo (IDT linker-1: 5’-rAppCTGTAGGCACCATCAATC-3’) using T4 RNA ligase 1 (New England Biolabs) without ATP. ~15-35 nt RNA captured by the 3’ linker oligo was purified using 8 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea, followed by a 5’ end ligation reaction using a DNA-RNA hybrid oligo (5’-ACGCTCTTCCGATCTrNrNrNrN-3’, rNrNrNrN as 4-nt barcode) using T4 RNA ligase 1 (New England Biolabs) with ATP. cDNA was synthesized by using SuperScript III Reverse transcriptase (Life Technologies) and primer SG460 (5‘-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’), followed by PCR amplification with primers SG-465 (5‘-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and SG-46X (5’-CAAGCAGAAGACGGCATACGAGAT[6-nt index]GTGACTGGAGTTCAGACGTGTGCTCTTCC-3’). To ensure that amplified DNA are still annealed to a true complement and avoid reannealing-distortion in the resulting libraries ( referenceParameswaran et al., 2007), PCR reactions were titrated with increasing cycle numbers; cycle numbers for which product levels have not saturated (i.e. product levels still able to increase substantially with additional cycles) were selected. After separating PCR products on 3 % agarose gels, DNA bands of the expected size were extracted (QIAchange® Gel Extraction Kit [Invitrogen]; omitting the 50°C heating step), followed by massive parallel DNA sequencing (Illumina HiSeq2000).
small RNA-seq (sample 18 and 19): Same as described in Gu SG et. al. 2012 (PMID: 22231482).
mRNA-seq (sample 13, 14, 15, 16, 17): 0.5-1 μg fragmented oligo(dT)-selected mRNA was used to construct each mRNA-seq library. The fragmented RNA was treated with T4 polynucleotide kinase (New England Biolabs). The 3’ end of RNA was ligated to an adenylated DNA oligo (IDT linker-1: 5’-rAppCTGTAGGCACCATCAATC-3’) using T4 RNA ligase 1 (New England Biolabs) without ATP. ~35-60 nt RNA captured by the 3’ linker oligo was purified using 8 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea, followed by a 5’ end ligation reaction using a DNA-RNA hybrid oligo (5’-ACGCTCTTCCGATCTrNrNrNrN-3’, rNrNrNrN as 4-nt barcode) using T4 RNA ligase 1 (New England Biolabs) with ATP. cDNA was synthesized by using SuperScript III Reverse transcriptase (Life Technologies) and primer SG460 (5‘-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’), followed by PCR amplification with primers SG-465 (5‘-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and SG-46X (5’-CAAGCAGAAGACGGCATACGAGAT[6-nt index]GTGACTGGAGTTCAGACGTGTGCTCTTCC-3’). To ensure that amplified DNA are still annealed to a true complement and avoid reannealing-distortion in the resulting libraries ( referenceParameswaran et al., 2007), PCR reactions were titrated with increasing cycle numbers; cycle numbers for which product levels have not saturated (i.e. product levels still able to increase substantially with additional cycles) were selected. After separating PCR products on 3 % agarose gels, DNA bands of the expected size were extracted (QIAchange® Gel Extraction Kit [Invitrogen]; omitting the 50°C heating step), followed by massive parallel DNA sequencing (Illumina HiSeq2000).
ChIP-seq for sample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21.
small RNA-seq (5' monophosphate-independent) for sample 11,12,18 and 19.
mRNA-seq for sample 13, 14, 15, 16,
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
~ 200 bp
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| Data processing |
Demultiplexing based on the inline barcode and index assignment. Remove 3' linker sequence for small RNA-seq and pre-mRNA-seq libraries. For small RNA-seq, mRNA-seq and pre-mRNA-seq, identical reads in each library were collapsed and counted as one read.
ChIP-seq reads were aligned to the C. elegans genome (version WS190/ce6) using Bowtie (version 0.12.7) with a command "bowtie C_elegans_genome -q -t -v 0 -a --strata --best -p 2 -5 3 -3 8 fastqFile bowtieOutput". RNA-seq reads were simliarly aligned to the C. elegans genome after collapsing identical reads in each library.
1kb-coverage values were calculated by counting the number of sequenced reads aligned to each 1kb window in the C. elegans genome. The numbers were then normalized by sequencing depth (in millions). A segment of 12-kb sequence at the end of chromosome I encodes C. elegans ribosomal RNAs. The actual copy numbers of the rRNA genes are larger than the copy numbers indicated by the annotated genome. This region was excluded for this analysis.
Genome_build: WS190/ce6
Supplementary_files_format_and_content: Sample 1-10, 20, and 21: matrix listing the whole-genome ChIP-seq coverage at 1-kb resolution. Column 1: chromosome; column 2: start position of the 1kb segment (zero-based) column 3 and subsequent columnes: RPKM values for different samples. Sample IDs were indicated in the first row.
Supplementary_files_format_and_content: Sample 11- 19: fasta files for RNA-seq reads with identical reads in each library collapsed to one read. The number of identical reads for each sequence was indicated as the last number in the sequence name. The inline barcodes in the 5' ends and the 3' linker sequence were removed.
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| Submission date |
May 28, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Sam Guoping Gu |
| E-mail(s) |
ggu@dls.rutgers.edu
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| Organization name |
Rutgers University
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| Department |
Molecular Biology and Biochemistry
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| Street address |
604 Allison Road
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| City |
Piscataway |
| State/province |
New Jersey |
| ZIP/Postal code |
08854 |
| Country |
USA |
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| Platform ID |
GPL13657 |
| Series (1) |
| GSE58031 |
Endogenous germline nuclear RNAi-mediated transcriptional silencing and histone H3 lysine 9 methylation in defined regions of the Caenorhabditis elegans genome |
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| Relations |
| BioSample |
SAMN02801803 |
| SRA |
SRX554723 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1399631_cov1kb_normalizedCov_H3K9me3_ChIP_WT_hrde1_copy_1.txt.gz |
570.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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