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| Status |
Public on Feb 01, 2016 |
| Title |
hrde-1_23C_G2_rep2_S2ChIP |
| Sample type |
SRA |
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| Source name |
Young adult whole animal
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| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype: hrde-1 (tm-1200)
temperature: 23˚C
generation: 2nd
repeat number: 2
antibody: Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2)
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| Growth protocol |
C. elegans strain N2 and hrde-1 (tm1200) were cultured on NGM plates with E. coli OP50 as the food source in a temperature controlled incubator. Worms were maintained at 15°C prior to the multigenerational temperature-shift experiment. For the multigenerational temperature-shift experiment, synchronized worms were cultured at 15°C for three generations, and then 23°C for six generations, followed by 15°C for three generations. Worms were synchronized by using the standard egg-prep protocol. L1s were released onto 10cm NGM OP50 plate (approximately 4500 L1s per plate) at each generation. Ten L1s from each generation were single picked for the brood size assay. For the 15°C generations (15C-G1-G3 and p15C-G1-G3), young gravid adults were collected 114-120 hours after L1s were released onto the OP50 plates. For the 23°C generations (23C-G1-G6), young gravid adults were collected 50-55 hours after L1s were released
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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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| Description |
DNA extracted from immunoprecipitated chromatin
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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 gene is calculated by the number of reads that perfectly aligned to both strands of the gene and 300 bp flanking sequence on each site normalized by the size of the gene and the sequencing depth of the library.
Genome_build: WS190/ce6
Supplementary_files_format_and_content: .xlsx file containing rpkm values of all genes for all 24 ChIP-seq libraries and 12 ChIP input libraries.
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| Submission date |
Oct 27, 2015 |
| 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) |
| GSE74399 |
Germline nuclear RNAi in C. elegans represses transgenerational inheritance of heat-induced gene transcriptional activation (chip-seq analysis) |
| GSE74405 |
Germline nuclear RNAi in C. elegans represses transgenerational inheritance of heat-induced gene transcriptional activation |
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| Relations |
| BioSample |
SAMN04218355 |
| SRA |
SRX1388763 |
| 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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