General Information
- Experiment ID
- OEX000045
- Experiment Name
- Fudan_ChIP_seq
- Experiment Type
- Genomic
- Description
- We examined Sp9 binding TF ChIP-seq from basal ganglionic eminence in E13.5 and E16.5 wide type mice.
- Protocol
- the E13.5 or E16.5 ganglionic eminences (including VZ, SVZ and MZ) (two litters for one independent ChIP experiment) were dissected, and fixed in 10 ml 1% formaldehyde for 10 min at room temperature. The formaldehyde was quenched by adding glycine to a final concentration of 0.125 M, and the samples were incubated for 5 min at room temperature. Tissues were then washed twice in cold PBS, pelleted, re-suspended in 1% SDS lysis buffer (with 1 mM PMSF/cocktail), and incubated on ice for 10 min. Sonication was performed using Bioruptor Pico (15-20 cycles) to keep most DNA fragments in the range of 200-500 bp. Eight micrograms anti-SP9 rabbit polyclonal antibody were incubated with the cleared chromatin (SDS concentration diluted to 0.1%) at 4°C overnight. Protein A+G beads were added to the samples and incubated for 4 h at 4°C. The samples were washed with low salt [150 mM NaCl, 0.1% w/v SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0)], high salt [500 mM NaCl, 0.1% w/v SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0)] and LiCl solutions [250 mM LiCl, 1% NP-40, 1% w/v sodium deoxycholate, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA], and the precipitated DNA was eluted in elution buffer (0.1 M NaHCO3 and 1% w/v SDS). The eluted DNA samples were reverse cross-linked overnight in the presence of 250 mM NaCl, treated with RNase and proteinase K and purified using the Zymo Research D5205 Kit. ChIP-seq sequencing libraries were prepared from both the input DNA and the precipitated DNA using the NEBNext DNA Library Prep Kit and Illumina standard adaptors, and were sequenced using a 150 bp pair-end strategy using the Illumina Hiseq 4000 platform.
Experiment information
- Attributes
-
library_layout Single library_selection ChIP library_strategy ChIP-Seq platform Illumina HiSeq 4000 - Project
-
1
Project ID Project name Create Date OEP000035 Sp8 and Sp9 Coordinately Promote Striatal Medium Spiny Neuron Production through Activating Six3 Expression 2018-05-24 - Samples
- 4
- Runs
- 4
Author Information
- Create Date
- 2018-05-24
- Last Modified
- 2019-12-03
- Submission
- hua feng
- PICB