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| Status |
Public on Jun 01, 2018 |
| Title |
H3K27me3_wt_ya_rep2 |
| Sample type |
SRA |
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| Source name |
young adults
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
antibody: H3K27me3
antibody id: 309-95209
antibody manufacturer: WAKO
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| Growth protocol |
Wild-type N2 were grown at 20°C in liquid culture to the adult stage using standard S-basal medium with HB101 bacteria, animals bleached to obtain embryos, and the embryos hatched without food in M9 buffer for 24 hrs at 20°C to obtain synchronized starved L1 larvae. L1 larvae were grown in a further liquid culture at 20°C to the desired stage, then collected, washed in M9, floated on sucrose, washed again in M9, then frozen into "popcorn" by dripping embryo or worm slurry into liquid nitrogen. Popcorn were stored at -80°C until use. Times of growth were L1 (4 hrs), L2 (20 hrs), L3 (30 hrs), L4 (45 hrs), young adults (60 hrs). Mixed populations of embryos were collected by bleaching cultures of synchronized one day old adults. glp-1(e2144) were raised at 15°C on standard NGM plates seeded with OP50 bacteria. Embryos were obtained by bleaching gravid adults and then approximately 6000 placed at 25°C on 150mm 2% NGM plates seeded with a 30X concentrated overnight culture of OP50. For harvest, worms were washed 3X in M9 and then worm slurry was frozen into popcorn by dripping into liquid nitrogen and stored at -80°C. Harvest times after embryo plating were D1/YA (53 hrs), D2 (71 hrs), D6 (167 hrs), D9 (239 hrs), D13 (335 hrs).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Frozen worm pellets were ground, fixed for 10 minutes in 1% formaldehyde, fixative was quenched with 125 mM glycine and cross-linked tissue washed 2X with PBS + protease inhibitors, then resuspended in FA buffer and subjected to sonication in a Bioruptor or Bioruptor pico to an average DNA size of 200bp. Extracts were spun down and the soluble fraction used for ChIP. For ChIPs of protein factors, 1mg of ChIP extract was incubated with 5ug antibody; for histone modifications, 500ug ChIP extract was incubated with 2ug of antibody. After overnight incubation with rotation at 4C, 40ul of equilibrated magnetic beads (coupled to protein A or G, depending on antibody) were added and incubated for 2 hrs at room temperature with rotation. Washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer were performed and DNA was eluted in elution buffer (1% SDS in TE with 250 mM NaCl) two times with 57 ul volume each, at 65°C. Samples were treated with RNAse, proteinase K and then crosslinks were reversed overnight at 65°C. DNA was purified on Qiagen PCR purification columns and used for ChIP library preparation.
ChIP or input DNA is blunt ended, A-tailed, ligated to adaptors, amplified by PCR, then size selected using AMPure beads. Illumina TruSeq adaptors and barcodes were used. Samples were sequenced on a HiSeq1500
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
ChIP-seq reads were aligned to the WS220 assembly of the C. elegans genome using bwa.
Low-quality (q<10), and (McMurchy et al., 2017)-blacklisted reads were discarded.
ChIP-seq data were normalized using the BEADS algorithm (Cheung et al., 2011).
ChIP-seq signal was either log-transformed (H3K4me3, H3K4me1) or z-scored (H3K36me3, H3K27me3).
Stage-specific coverage was calculated by averaging across biological replicates, and smoothed by binning the signal at 10bp resolution.
Genome_build: WBcel215/ce10 (WS220)
Supplementary_files_format_and_content: Stage-specific normalized ChIP-seq profiles of histone marks.
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| Submission date |
May 14, 2018 |
| Last update date |
Jun 01, 2018 |
| Contact name |
Julie Ahringer |
| E-mail(s) |
ja219@cam.ac.uk
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| Organization name |
University of Cambridge
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| Department |
The Gurdon Institute
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| Street address |
Tennis Court Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
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| Platform ID |
GPL18730 |
| Series (2) |
| GSE114440 |
Chromatin accessibility dynamics across C. elegans development and ageing [ChIP-seq] |
| GSE114494 |
Chromatin accessibility dynamics across C. elegans development and ageing |
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| Relations |
| BioSample |
SAMN09207943 |
| SRA |
SRX4082381 |
| 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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