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| Status |
Public on Nov 28, 2013 |
| Title |
H3K27me3_neurula |
| Sample type |
SRA |
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| Source name |
whole embryo
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| Organism |
Xenopus tropicalis |
| Characteristics |
cell type: whole embryo
developmental stage: neurula (stage 16)
chip antibody: H3K27me3 (Upstate/Millipore, 07-449)
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| Growth protocol |
Xenopus tropicalis embryos were obtained from a natural mating procedure after human chorion gonadotropin injection, dejellied in 3% cysteine and collected at the indicated stage.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Embryos (n=300-600) were fixed in 1% formaldehyde for 30 minutes at the indicated developmental stage. Embryos were subsequently washed in 125 mM glycine / 25% Marc's modified Ringer's medium (MMR) and twice in 25% MMR, homogenized in sonication buffer (20 mM Tris•HCl, pH 8/10 mM KCl/1mM EDTA/10% glycerol/5 mM DTT/0.125% Nonidet P-40, and protease inhibitors), sonicated using a Bioruptor sonicator (Diagenode), centrifuged, and supernatants were frozen. ChIP extract was diluted with IP buffer (50 mM Tris•HCl, pH 8/100 mM NaCl/2mM EDTA/1 mM DTT/1% Nonidet P-40, and protease inhibitors) and incubated with 1-5 µg of antibody; 12.5 µl Prot A/G beads (Santa Cruz) was added and incubated for 15 hours on a rotating wheel at 4°C. The beads were subsequently washed in ChIP1 buffer (IP buffer plus 0.1% sodium deoxycholate), ChIP2 buffer (ChIP1 buffer with 500 mM NaCl final concentration), ChIP3 buffer (ChIP1 buffer with 250 mM LiCl), ChIP1 buffer, and TE buffer (10 mM Tris, pH 8/1 mM EDTA). The material was eluted in 1% SDS in sodium carbonate, cross-linking was reversed by adding 16 µl NaCl and incubating at 65°C for 5 h, DNA was phenol extracted and precipitated. The following antibodies were used: α-H3K4me3 (Abcam ab8580), α-H3K27me3 : (Upstate/Millipore 07-449), α-H3K4me1 (Diagenode CS-037-100), Jarid2 (Abcam ab48137), EZH2 (Active Motif 39103), RNAPII (Diagenode AC-055-100). For all ChIP-seq samples three independent biological replicates of difference chromatin isolations were pooled.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Basecalling was performed using Illumina CASAVA
Filtering was performed using the default Illumina Chastity Filter
Read were aligned to the Xenopus tropicalis genome (version JGI 7.1) using bwa
Genome_build: JGI 7.1
Supplementary_files_format_and_content: BED alignment files were generated from the alignments produced by bwa, duplicates were marked using bamUtil version 1.0.2 and filtered out, all reads mapping to multiple genomic positions were filtered out; _peaks.bed files were produced from the PeakRanger summit file and post-processed to show both region and summit while still being valid BED format, region coordinates are present in column 2 and 3, summit coordinates are present in columns 7 and 8 using the thickStart and thickEnd properties of the BED format; wig files were generated by normalizing experiments between developmental stages per antibody by random removal of reads, extending the reads in the BED file to 300bp and calculating the average number of reads in 10 bp windows.
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| Submission date |
Sep 26, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Gert Jan Veenstra |
| E-mail(s) |
g.veenstra@science.ru.nl
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| Phone |
+31 24 3610541
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| Organization name |
Radboud University
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| Department |
Molecular Developmental Biology
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| Street address |
Geert Grooteplein 28
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL13741 |
| Series (1) |
| GSE41161 |
Principles of nucleation of H3K27 methylation during embryonic development |
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| Relations |
| SRA |
SRX189706 |
| BioSample |
SAMN01731017 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1009595_H3K27me3_neurula.bed.gz |
57.7 Mb |
(ftp)(http) |
BED |
| GSM1009595_H3K27me3_neurula.wig.gz |
12.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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