|
| Status |
Public on Nov 04, 2015 |
| Title |
Input for ChIP-Seq in mouse neural stem cells |
| Sample type |
SRA |
| |
|
| Source name |
cerebral cortex
|
| Organism |
Mus musculus |
| Characteristics |
cell line: CD-1
tissue: cerebral cortex
cell type: Mouse neural stem cell
|
| Growth protocol |
Mouse neural stem cells were cultured and passaged every 2 days as monolayers in MEM/F12 medium containing Glutamax, non-essential amino acids, B27, N2 supplement, 20 ng/ml EGF, and 20 ng/ml FGF. Cells were dissociated using Accutase (Invitrogen) and seeded onto poly-d-lysine-coated plates or dishes.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed using 107 mouse neural stem cells per reaction. Cells were dissociated by treatment with Accutase and cross-linked in 1% (vol/vol) formaldehyde for 10 min at RT with rotation. Then, 0.125 M glycine was used to quench the cross-linking reaction. Cells were pelleted and washed with ice-cold PBS. Next, nuclei were isolated and a Bioruptor sonicator (Diagenode) was used to shear chromatin DNA. Either 5 ml of OLIG2 antibody (AB9610, Chemicon) or 5 ml of normal rabbit IgG (#2729, Cell Signaling) was added to Dynabeads Protein A (Invitrogen) beads and incubated for 3 h at 4 °C with rotation. Then, the Dynabeads-antibody complexes were incubated with sheared chromatin DNA overnight at 4 °C. After immunoprecipitation, the precipitated complex was treated with RNase A and Proteinase K, and incubated at 65 °C overnight to reverse crosslinks.
The ChIP-Seq library was constructed by using DNA SMART ChIP-Seq Kit according to the manufacturer's instructions (Clontech) and was sequenced on the Illumina HiSeq 2000 Sequencer.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
The generated reads were mapped to the mouse genome mm10 using Bowtie version 0.12.7 and MACS version 1.4.2 were used to call peaks for ChIP-Seq data
The output from MACS was filtered using the following more stringent criteria: (1) p value cutoff <10−9; (2) fold_enrichment > 5-fold (3) tag number >20.
Genome_build: mm10
Supplementary_files_format_and_content: Excel file include peaks(OLIG2 binding site) call by MACS
|
| |
|
| Submission date |
Nov 03, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Xiaomin Dong |
| E-mail(s) |
xiaomin.dong@yahoo.com
|
| Organization name |
UT health
|
| Department |
Department of neurosurgery
|
| Lab |
Dr.Jiaqian Wu
|
| Street address |
1825 Pressler Street
|
| City |
houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE74646 |
Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination (OLIG2 Chromatin immunoprecipitation sequencing (ChIP-Seq) in mouse neural stem cells) |
| GSE74648 |
Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination |
|
| Relations |
| BioSample |
SAMN04233139 |
| SRA |
SRX1411157 |
