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| Status |
Public on Jul 23, 2018 |
| Title |
H3K4me3 early embryo ChIP, rep2 |
| Sample type |
SRA |
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| Source name |
Mixed stage early embryos
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain and genotype: N2 wild type
purity of sample: 1
chip antibody: H3K4me3 (Wako, MABI0304, #305-34819)
xchromosome: autosome ratio: X:A=1:1 (vast majoriy XX hermaphrodites)
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| Growth protocol |
Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture at 20oC. Live embryos were cross-linked in M9 + 1.85% formaldehyde for 30 minutes at room temperature. Embryos were then washed twice with M9 Buffer, once with 100 mM Tris-HCl pH 7.5 and once with FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl). Pellets were frozen at -80C. For a detailed protocol see http://www.modencode.org/.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl) + protease inhibitors. Sample was dounced 30 times using tight pestle at 4ºC, then sonicated 9 times on ice at the following settings: 30 sec; power output 3; 70% duty cycle. Cell debris was removed by centrifuging at 13,000 rpm for 15 minutes at 4ºC. The supernatant was filtered through a Millipore Ultrafree-MC 0.45 um filter unit (cat. UFC30HV0S) at 13,000 rpm, 4°C for 1 minute to remove lipids. Protein concentration was determined, and the extract was aliquoted and stored at -80ºC. For a detailed protocol see http://www.modencode.org/.
DNA was incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends, then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3' ends (a single A overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments were ligated with single-end 'Homebrew' adaptors that contained an index sequence within. After ligation, the samples were purified twice using SPRI beads, allowing for a size selection step to get rid of excess adaptors. Samples were then amplified by PCR with single-end primers. Depending on the original fragmentation of DNA, samples were purified by SPRI or by separation and purification from an agarose gel.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ul of IgG dynabeads (Dyna), and washed 5 minutes with 1.5 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 uL elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Zymo kit. For a detailed protocol see http://www.modencode.org/.
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| Data processing |
Raw sequence reads from the Illumina HiSeq (50 bp single-end read sequencing for ChIP-seq) were mapped to the C. elegans genome (Wormbase version WS220) using Bowtie with default settings.
MACS2 was used to call peaks and to create bedgraph files with the following settings: -g ce --bdg --keep-dup=auto --broad --broad-cutoff=0.01 --nomodel --extsize=250.
Custom scripts were used to scale bedgraph files to 10 million total autosomal reads (to account for different X:A ratios of oocytes and embryo samples compared to sperm samples) and converted to bigwig using the bedGraphToBigWig UCSC Genome Browser tool.
Genome_build: WS220
Supplementary_files_format_and_content: bigWig containing scaled read coverage
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| Submission date |
Jun 12, 2018 |
| Last update date |
Jul 24, 2018 |
| Contact name |
Susan Strome |
| E-mail(s) |
sstrome@ucsc.edu
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| Organization name |
UC Santa Cruz
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| Department |
MCD Biology
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| Street address |
1156 High St
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| City |
Santa Cruz |
| State/province |
CA |
| ZIP/Postal code |
95064 |
| Country |
USA |
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| Platform ID |
GPL9309 |
| Series (2) |
| GSE115704 |
Distribution of histone modifications across the genome in C. elegans sperm vs. oocytes vs. early embryos |
| GSE115709 |
Epigenome of C. elegans sperm, oocytes, and early embryos (MNase-seq, ChIP-seq, RNA-seq) |
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| Relations |
| BioSample |
SAMN09402982 |
| SRA |
SRX4200536 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3187958_H3K4me3.Eembryo14.rep2_s_1_seq_scaled.10Mil.Autos.bw |
9.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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