|
| Status |
Public on Aug 04, 2022 |
| Title |
PolII_KCl_60min |
| Sample type |
SRA |
| |
|
| Source name |
E15.5 C57BL/6 embryonic mouse cortices
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: E15.5
cell type: primary corticol neurons
chip antibody used for chip: Abcam ab817(Lot#GR313984-14)
|
| Treatment protocol |
For ChIP-seq experiments, mouse cortical neurons were plated at an approximate density of 2 × 10^6 on 35-mm dishes. Neurons were plated in 2 mL neuronal medium. One ml of the medium was replaced with 1 ml fresh warm medium on Day3 and Day6. Prior to KCl depolarization, neurons were silenced with 1 μM tetrodotoxin on Day6 (TTX; Fisher). Neurons were subsequently stimulated on Day7 by adding warmed KCl depolarization buffer (170 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) directly to the neuronal culture to a final concentration of 31% in the neuronal culture medium within the culture plate or well for the indicated time.
|
| Growth protocol |
Neurons were grown in neuronal medium consisting of Neurobasal medium containing B27 supplement (2%; Thermo Fisher), penicillin-streptomycin (50 g/mL) and Glutamax (1x; Thermo Fisher). Neurons were subsequently plated and placed in a cell culture incubator that maintained a temperature of 37 °C and a CO2 concentration of 5%. Ten minutes after plating neurons, the medium was completely aspirated from cells and replaced with fresh warm neuronal medium. Neurons were grown in vitro until Day7.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with 1% formaldehyde at room temperature for 10 min. And then the cross-linking was quenched with 0.125M glycine for 5 min. Chromatin was fragmented using a Bioruptor® Pico (Diagenode) for 10 min at high power, with an interval of 30 s between pulses to get around 200bp fragments and precleared using 20 μl Protein G Dynabeads (Life Technologies, Cat# 10009D). Subsequently, the soluble chromatin was incubated with 2-5 μg antibodies at 4°C overnight. Immunoprecipitated complexes were collected using 30 μl Protein G Dynabeads, which have been saturated with PBS/1% BSA over night at 4°C, per reaction. For all ChIPs, after de-crosslinking overnight at 65°C, final ChIP DNA was extracted and purified using QIAquick spin columns (QIAGEN).
The libraries were constructed following Illumina’s Chip-Seq Sample prep kit.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.4
Reads were aligned to the mm10 genome assembly using Bowtie2.
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph
|
| |
|
| Submission date |
Oct 11, 2021 |
| Last update date |
Aug 04, 2022 |
| Contact name |
Yuliang Tan |
| E-mail(s) |
yut020@ucsd.edu
|
| Organization name |
University of California, San Diego
|
| Department |
School of Medicine
|
| Street address |
Room 345, CMM West, 9500 Gilman Drive, Mail Code 0648
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037-0648 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE135808 |
Topoisomerase 1 Nicking-dependent HP1gamma Engagement Mediates Acute Activation of Phase-separated Enhancers |
| GSE185648 |
Ku70/HP1g are presented at the KCl induced acutely activated enhancers [ChIP-seq] |
|
| Relations |
| BioSample |
SAMN22213258 |
| SRA |
SRX12569169 |
