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| Status |
Public on Jan 01, 2017 |
| Title |
Pol II_ChIP-seq_met-2set-25_GFP_RNAi |
| Sample type |
SRA |
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| Source name |
Young adult whole animal
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| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype/variation: met-2(n4256);set-25(ok5021)
temperature: 19˚C
feeding: GFP RNAi
chip antibody: Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2)
molecule subtype: DNA extracted from immunoprecipitated chromatin
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| Growth protocol |
C. elegans strains N2, hrde-1 (tm1200), set-32(ok1457), met-2(n4256);set-25(ok5021), and met-2(n4256);set-25(ok5021);set-32(ok1457) were cultured on NGM plates with E. coli OP50 or oma-1 RNAi or GFP RNAi as the food source in a temperature controlled incubator at 19°C for all experiments. For OP50 ChIP-seq experiments synchronized worms were raised at 19°C and young adult worm samples were collected. Worms were synchronized by using the standard egg-prep protocol. For oma-1 and GFP RNAi experiments worms were raised at 19°C on NGM plates with corresponding RNAi feeding for ~3 generations and until majority of worms reached gravid adult stage. These worms were synchronized and L1 (~4500 worms per plate) worms were released on the corresponding RNAi feeding NGM plates, raised at 19°C and young adults worm samples were collected.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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) or anti-Pol II-S2 (ab5095, Abcam) 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 was treated similarly to reverse crosslink) and then subject to organic extraction and precipitation of DNA and RNA.
DNA libraries were prepared using KAPA Hyper Prep Kits (KAPA Biosystems) according to the manufacturer's instructions
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
50 nt Illumina sequencing reads were aligned to the C. elegans genome using bowtie[0.12.7].
rpkm value for each 1kb is calculated by the number of reads that perfectly aligned to both strands of 1kb-non-overlaping regions, normalized by the sequencing depth of the library in million.
Genome_build: WS190/ce6
Supplementary_files_format_and_content: .xlsx file containing rpkm values of all 1kb regions for 15 H3K9me3 Chip-seq, 10 Pol II (S2) ChIP-seq libraries. H3K9me3 ChIP-seq results of OP50 feeding experiments were used as replica 1 to identify different HMT-dependent K9 targets replica 1. H3K9me3 ChIP-seq results from RNAi control experiment (GFP RNAi) were used as replica 2 to identify HMT-depende.xlsx file containing rpkm values of all 1kb regions for 15 H3K9me3 Chip-seq, 10 Pol II (S2) ChIP-seq libraries. GRH and GRTS regions are previously defined germline nuclear RNAi-dependent heterochromatin (GRH) and transcriptional silencing (GRTS) targets.H3K9me3 ChIP-seq results of OP50 feeding experiments were used as replica 1 to identify different HMT-dependent K9 targets replica 1. H3K9me3 ChIP-seq results from RNAi control experiment (GFP RNAi) were used as replica 2 to identify HMT-dependent K9 targets replica 2. All replica HMT-dependent K9 target lists are reported in the table. In addition, miss-annotated regions are also reported in the table. Whole genome classification of H3K9me3 level is also labeled in the table as K9 tire: 1-3 correspond to the top 5% H3K9me3 regions, 4 correspond to the next 5-25%, tire 5 is 25-50%, tire 6 is 50-75%, tire 7 is 75-100% (bottom H3K9me3 quartile, correspond to the background level) excluding tire 8 - extreme low values (=0 or ~0). nt K9 targets replica 2. All replica HMT-dependent K9 target lists are reported in the table. In addition, miss-annotated regions are also reported in the table. Whole genome classification of H3K9me3 level is also labeled in the table as K9 tire: 1-3 correspond to the top 5% H3K9me3 regions, 4 correspond to the next 5-25%, tire 5 is 25-50%, tire 6 is 50-75%, tire 7 is 75-100% (bottom H3K9me3 quartile, correspond to the background level) excluding tire 8 - extreme low values (=0 or ~0).
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| Submission date |
Sep 07, 2016 |
| 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 (2) |
| GSE86513 |
Decoupling the downstream effects of germline nuclear RNAi reveals that transcriptional repression and heritable RNAi are independent of the H3K9me3 response in C. elegans [ChIP-seq] |
| GSE86517 |
Decoupling the downstream effects of germline nuclear RNAi reveals that transcriptional repression and heritable RNAi are independent of the H3K9me3 response in C. elegans |
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| Relations |
| BioSample |
SAMN05736309 |
| SRA |
SRX2144182 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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