 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 07, 2016 |
| Title |
HH14_H3K4me3_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Spinal neural tube (brachial level)
|
| Organism |
Gallus gallus |
| Characteristics |
strain: White Leghorn
tissue: Spinal neural tube (brachial level)
developmental stage: HH14
genotype: WT
chip antibody: H3K4me3 (39159, Active Motif)
|
| Treatment protocol |
Not applicable
|
| Growth protocol |
Fertilized chicken eggs (White Leghorn, Gallus gallus domesticus) were obtained from a local breeder (LSL Rhein-Main, Dieburg). Incubation was done at 37°C and 80% humidity in a normal poultry egg incubator (Typenreihe Thermo-de-Lux). After following microsurgical procedures, the eggs were re-incubated until the embryos reached the desired developmental stages. The developmental progress was determined according to the staging system of Hamburger Hamilton (HH) (Hamburger and Hamilton, 1992).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fertile chicken eggs were incubated at 37°C for 48 hrs until they reached stage HH14(Hamburger and Hamilton, 1992). Embryos were isolated from the eggs, transferred into 20 ml of 1xPBS and the extraembryonic membranes were removed. Under the stereomicroscope, the transverse SNT sections from chicken embryos were collected at the brachial level, extending caudally into the thoracic region (Figure 1C). Most of the tissues that are located laterally/ventrally to the neural tube (e.g. lateral plate mesoderm, endoderm and aorta) were removed manually. Each section was immersed in 90 mm petri dish, containing 3 ml of warm (37°C) trypsin (Trypsin.EDTA solution 1:250 (IX), GIBCO) (Figure 1C). Trypsinization time was tightly controlled to avoid over-digestion and dispersion of neural tissue and to retain the structural integrity of the SNT. Once connective tissues became loosened near the outline of the neural tube, which is easily detectable under a stereomicroscope, trypsin was inactivated by rinsing with cold (4°C) DMEM containing 10% FBS and samples were transferred into cold 1 x PBS. Following trypsin treatment, mesenchymal and ectodermal tissues (e.g. paraxial mesoderm, notochord, surface ectoderm) were removed manually using forceps. After isolation, SNT fragments were pooled in a 1.5 ml tube, flash-frozen in liquid nitrogen and stored at -80°C. For each ChIP-seq experiment, ~25-30 SNT sections isolated from stage HH14 chicken embryos were used. Dissected neural tubes were briefly homogenized in DMEM media with 10% FBS serum/1M sodium butyrate and cross-linked with 1% formaldehyde at room temperature with rotation for 15 mins. Cross-linking was quenched with 0.125 M glycine and neural tissue was rinsed once with cold 1 X PBS and resuspended in 300 µl of sonication buffer (10mM Tris, 100mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate and 0.5% N-lauroylsarcosine) and incubated at 4°C for 10 mins. Chromatin was then sonicated using Bioruptor plus (Diagenode) with high level settings for 11 cycles with 30-sec pulses at 4 °C (45-sec pause between pulses) to generate DNA fragments of ~200-500 bp. 30 µl of the sonicated chromatin was kept as input DNA, while the remaining chromatin was incubated overnight with 5µg of antibodies against H3K27me3 (39536, Active Motif), H3K27ac (Active Motif) or H3K4me3 (39159, Active Motif)(Rada-Iglesias et al., 2011), followed by 4-6 hours incubation with Protein G Dynabeads (Life Technologies). Beads were washed 4 times with 1ml of cold RIPA washing buffer (50mM Hepes, 500mM LiCl, 1mM EDTA, 1% NP-40 and 0.7% Na-Deoxycholate) and once with Tris/EDTA buffer (TE) containing 50mM NaCl. Immunoprecipitated chromatin was eluted from the beads by adding elution buffer (50mM Tris, 10mM EDTA and 1% SDS) and vortexing at 65°C for 15 mins. Crosslinking was reversed by incubating the eluted samples at 65°C overnight. Samples were then diluted (1:1) with TE and treated with RNase A (0.2µg/µl) for 90 mins at 37°C, followed by proteinase K (0.2µg/µl) treatment at 50°C for 90 mins. DNA was purified using Phenol:Chloroform, followed by ethanol precipitation and resuspended in water.
DNA libraries were prepared using the Illumina TruSeq RNA sample preparation Kit v2 starting from the end repair step of the protocol. Up to 100 ng ChIP DNA was used as starting material. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified for 18 PCR cycles to create the final libraries.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Basic read quality check was performed using FastQC (Babraham Bioinformatics) and read statistics were obtained with SAMtools
Reads were mapped to the chicken reference assembly, version galGal4, using BWA
Normalized bedGraph files were generated using DeepTools
Peaks were called using MACS2
Genome_build: galGal4
|
| |
|
| Submission date |
Nov 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Milos Nikolic |
| E-mail(s) |
milosn@gmail.com
|
| Organization name |
Center for Molecular Medicine Cologne
|
| Street address |
Robert Koch Str. 21
|
| City |
Koeln |
| State/province |
Nordrhein-Westfalen |
| ZIP/Postal code |
50674 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19005 |
| Series (2) |
| GSE89604 |
Epigenomic-based identification of major cell identity regulators within heterogeneous cell populations [ChIP-seq] |
| GSE89606 |
Epigenomic-based identification of major cell identity regulators within heterogeneous cell populations |
|
| Relations |
| BioSample |
SAMN05990952 |
| SRA |
SRX2333009 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2385293_HH14_H3K4me3_rep2_1x_bg.bedgraph.gz |
174.5 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
