|
| Status |
Public on Oct 20, 2020 |
| Title |
HA.Ascl2_HA_ChIP_B |
| Sample type |
SRA |
| |
|
| Source name |
Oligodendrocyte progenitor cells (OPCs)
|
| Organism |
Mus musculus |
| Characteristics |
cell type: epiblast stem cell derived OPCs
antibody: HA (Cell Signaling Technologies, 3724S, 15ug)
number of cells per chip: 60 million OPCs
|
| Treatment protocol |
Comparison of HA pulldown between control and HA-Ascl2 OPCs.
|
| Growth protocol |
OPCs were grown in DMEM F12 supplemented with N2max and B27 and Fgf2 and Pdgfa.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei isolation and chromatin shearing were performed using the Covaris TruChIP protocol following manufacturer’s instructions for the “high-cell” format. In brief, 5-20 million cells were crosslinked in “Fixing buffer A” supplemented with 1% fresh formaldehyde for 10 minutes at room temperature with oscillation and quenched for 5 minutes with “Quench buffer E.” These cells were then washed with PBS and either snap frozen and stored at -80, or immediately continued to nuclei extraction and shearing per the manufacturer protocol. The samples were sonicated with the Covaris S2 using the following settings: 5% Duty factor 4 intensity for four 60-second cycles. Sheared chromatin was cleared and incubated overnight at 4 degrees with antibody that was pre-incubated with protein G magnetic DynaBeads (ThermoFisher).
Protein G dynabeads were then washed, eluted, reverse cross-linked and treated with RNase A followed by proteinase K. ChIP DNA was purified using Ampure XP beads (Aline Biosciences, C-1003-5) and then used to prepare illumina sequencing libraries.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
HA-Ascl2 OPCs
|
| Data processing |
Reads were quality and adapter trimmed using Trim Galore! Version 0.4.1.
Trimmed reads were alighed to mm10 with Bowtie2 version 2.3.2.
Duplicate reads (potential artifacts of PCR in library preparation) were removed using Picard MarkDuplicates.
Genome_build: mm10
Supplementary_files_format_and_content: bigWIG files were generated using Deeptools bamCoverage with reads per genome coverage normalization. BED files in a narrowPeaks format from MACS2.
|
| |
|
| Submission date |
Aug 11, 2020 |
| Last update date |
Oct 20, 2020 |
| Contact name |
Paul Tesar |
| E-mail(s) |
paul.tesar@case.edu
|
| Organization name |
Case Western Reserve University
|
| Department |
Genetics & Genome Sciences
|
| Street address |
10900 Euclid Ave.
|
| City |
Cleveland |
| State/province |
Ohio |
| ZIP/Postal code |
44106 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE143472 |
Non-Canonical Targets of HIF1a Impair Oligodendrocyte Progenitor Cell Function [ChIP-Seq] |
| GSE143474 |
Non-Canonical Targets of HIF1a Impair Oligodendrocyte Progenitor Cell Function |
|
| Relations |
| BioSample |
SAMN15793475 |
| SRA |
SRX8926860 |
