|
| Status |
Public on Apr 19, 2024 |
| Title |
ChIPWT H3K4me1 Ectoderm Rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
Early Embryo
|
| Organism |
Xenopus tropicalis |
| Characteristics |
tissue type: Animal Cap (Ectoderm)
developmental stage: Nieuwkoop Faber 10.25
antibody: H3K4me1
clock time (hours post-fertilization): 7
agent: untreated
|
| Growth protocol |
Xenopus tropicalis females were injected with 10 units of Chorulon (Merck and Co.) 1-3 nights before embryo collection and 100 units of Chorulon on the day of embryo collection. Eggs were collected in a dish coated with 0.1% BSA in 1/9x MMR. The eggs are in vitro fertilized with sperm suspension in 0.1% BSA in 1/9x MMR. The embryos are dejellied with 3% cysteine in 1/9x MMR, pH 7.8. After 10 minutes, embryos are washed with 1/9x MMR and transferred to 1/9x MMR.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Samples were dissected from 100 fixed embryos and were then homogenized in RIPA buffer and incubated on ice for 10 minutes. Samples were then centrifuged at 14,000rpm for 10 minutes at 4C. Pellets were resuspended in RIPA buffer and sonicated on ice to an average size of 100-500bp. The resulting samples were centrifuged at 14,000 rpm for 30 minutes at 4C to remove insoluble cellular debris. The sheared chromatin was pre-cleared by incubating with 20 ¼L Protein G-coated Dynabeads (Invitrogen) for 2 hours at 4C with rotation. Meanwhile, another 20 ¼L Protein G Dynabeads were incubated in a tube rotator in 1 mg/mL torula RNA and 0.5% BSA in 1x PBS for 30 minutes at 4C to block non-specific binding. This second 20 ¼L Protein G Dynabeads were then pre-bound with 3 ug anti-H3K4me1 antibody at 4C for > 1 hour. The pre-cleared chromatin was added to antibody-bound Dynabeads, and incubated overnight at 4¡C on a tube rotator. The next day, the beads were washed, and DNA was eluted off the beads with TE buffer containing 1% SDS, and reverse-crosslinked at 65C overnight. Sonicated input control was diluted 3-fold with elution buffer and also incubated at 65C. All samples were treated with RNAse A, Proteinase K, phenol/chloroform extracted, and ethanol precipitated overnight. DNA pellets were resuspended in Qiagen EB solution.
Library Construction Protocol: Nextflex ChIP-seq kit (Bioo Scientific)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
ChIPWT-h3k4me1-ecto_peaks.narrowPeak.gz
|
| Data processing |
H3K4me1 ChIP-seq reads from biological replicates were concatenated and then aligned to the X. tropicalis genome v9.0 (Mitros et al., 2019; Karimi et al., 2018) using Bowtie v2.2.7 (Langmead and Salzberg 2012) with default options and peaks were called against stage 10 input DNA (Charney et al., 2017) using Macs v2.0.10 (Zhang et al., 2008) with the option –p 0.0000000001 but otherwise default options.
Genome_build: Xenopus tropicalis v9.0
Supplementary_files_format_and_content: ChIP-Seq: NarrowPeak (BED)
|
| |
|
| Submission date |
Apr 20, 2021 |
| Last update date |
Apr 19, 2024 |
| Contact name |
Kitt D. Paraiso |
| E-mail(s) |
kparaiso@uci.edu
|
| Phone |
949-824-7950
|
| Organization name |
University of California, Irvine
|
| Department |
Department of Developmental and Cell Biology
|
| Lab |
Ken W.Y. Cho
|
| Street address |
4410 Nat Sci II, University of California, Irvine
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92697 |
| Country |
USA |
| |
|
| Platform ID |
GPL23182 |
| Series (1) |
| GSE172395 |
Maternal and zygotic factors sequentially shape the tissue regionalization of chromatin landscapes in early vertebrate embryos |
|
| Relations |
| BioSample |
SAMN18808492 |
| SRA |
SRX10641450 |
