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| Status |
Public on Nov 06, 2024 |
| Title |
hNSC, CHD8, ChIP |
| Sample type |
SRA |
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| Source name |
hNSCs
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| Organism |
Homo sapiens |
| Characteristics |
cell line: hNSCs
cell type: H9 human embryonic stem cell-derived neural stem cells
genotype: WT
chip antibody: CHD8 (Bethyl, A301-225A)
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| Growth protocol |
hNSCs were cultured on Geltrex-coated dishes (Geltrex LDEV-free reduced growth factor membrane matrix, A1413202, Thermo Fisher Scientific) in KnockOut DMEM/F12 (12660012, Invitrogen) supplemented with 2 mM L-Glutamine (25030024, Thermo Fisher Scientific), 20 ng/ml EGF (315-09, Peprotech), 20 ng/ml FGF (100-18B, Peprotech) and 2% of StemPro Neural Supplement (A1050801, Thermo Fisher Scientific)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
hNSCs were double crosslinked by incubation with 2 mM disuccinimidyl glutarate solution, followed by an incubation with 1% of buffered formaldehyde solution. Subsequently, the cell pellets were lysed with lysis buffer (1% SDS, 50 mM TrisHCl pH 8.1, 10mM EDTA pH 8.0 and 1x CEF protease inhibitors (Roche)) and sonicated using a Bioruptor Pico sonication device (Diagenode, B01060001) for 14 cycles of 30sec on and 30sec off. The ChIP was performed on the resulting sheared chromatin by incubation overnight at 4°C with ProtG Dynabeads (Invitrogen, #100-04D) that had been pre-coupled to either the CHD8 antibody, TRRAP antibody or IgG antibody (Diagenode, C15410206) as control. Following the overnight incubation, the Dynabeads were washed twice with Low salt wash buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl), once with High salt wash buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% Triton X-100, 500 mM NaCl), once with LiCl wash buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25 M LiCl, 0.5% IGEPAL, 1% NaDeoxycholate) and once with T10E1 (10 mM TrisHCl pH 8.0, 1 mM EDTA). The bound complexes were eluted of the beads by incubation for 1 hour at 65°C in ChIP elution buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS) with occasional gentle vortexing. The samples were de-crosslinked overnight by incubation at 55°C. DNA was extracted from the elutions by the Phenol:Chloroform:Isoamyl alcohol (PCI) method and diluted in water.
The DNA libraries for the input, IgG, CHD8 and TRRAP ChIP samples were prepared using the ThruPLEX V2 DNA sample preparation protocol from Takara Bio.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
I20-1332-03
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| Data processing |
The reads were first trimmed by removing the Illumina adapters, followed by alignment to the reference genome GRCh38 using HISAT2.
After alignment, low-quality, duplicated fragments and fragments that exceeded 150 bases in length were removed.
Peaks were called using MACS2 (version 2.1.1.20160309) with FDR thresholds 1e-4 (CHD8) and 1e-3 (TRRAP).
Peaks overlapping with ENCODE Blacklist have been removed using GenomicRanges package v.1.48 in R. Subsequently, the peaks longer than 1kb have been filtered as they are mostly artefacts not presenting narrow peaks for TF binding.
Assembly: GRCh38
Supplementary files format and content: BigWig files: fragment coverage after filtering
Supplementary files format and content: BED files: peaks after filtering
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| Submission date |
Nov 06, 2023 |
| Last update date |
Nov 06, 2024 |
| Contact name |
Raymond A Poot |
| E-mail(s) |
r.poot@erasmusmc.nl
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| Organization name |
Erasmus MC
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| Department |
Cell Biology
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| Street address |
Wytemaweg 80
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| City |
Rotterdam |
| ZIP/Postal code |
3015 GE |
| Country |
Netherlands |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE247132 |
A CHD8-TRRAP axis facilitates MYC and E2F target gene regulation in human neural stem cells. [ChIP-seq] |
| GSE247133 |
A CHD8-TRRAP axis facilitates MYC and E2F target gene regulation in human neural stem cells. |
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| Relations |
| BioSample |
SAMN38123150 |
| SRA |
SRX22385643 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7884007_CHD8_FDR_0_0001.bed.gz |
376.3 Kb |
(ftp)(http) |
BED |
| GSM7884007_I20-1332-03-CHD8.bw |
126.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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