NextSeq 500 sequencing; ChIP-seq of CIC (Capicua) and histone marks (H3K9ac, H3K27ac and H3K4me1) in glioma neural stem cells (GNS) G144, human foetal neural stem cells (hNSC) and mouse embryonic stem cells (mESC),
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Neural
Cell type
G144
NA
NA
Attributes by original data submitter
Sample
ENA first public
2018-05-30
ENA last update
2018-04-24
ENA-CHECKLIST
ERC000011
External Id
SAMEA4608337
INSDC center alias
BRIC - Biotech Research and Innovation Centre
INSDC center name
BRIC - Biotech Research and Innovation Centre
INSDC first public
2018-05-30T17:02:00Z
INSDC last update
2018-04-24T13:06:01Z
INSDC status
public
Submitter Id
E-MTAB-6682:G144_WT_4h_CIC
broker name
ArrayExpress
cell line
G144
cell type
glial cell
common name
human
developmental stage
adult
disease
glioblastoma multiforme
genotype
wild type genotype
organism part
brain
sample name
E-MTAB-6682:G144_WT_4h_CIC
Sequenced DNA Library
library_name
G144_WT_4h_CIC_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
CRISPR mediated KO of CIC: sgRNAs (Target sequence: mESCs: GCCTTCATGATCTTCAGCAAG; G144: GGGCGAGTGGTGGTATGCCC) were cloned into pSpCas9(BB)-2A-GFP (Addgene 48138) and transfected into mESCs (Lipofectamine 2000) or G144 cells (Amaxa Nucleofector II, Program A033). GFP-positive cells were single cell sorted 48h post-transfection, expanded and screened by immunoblotting for CIC deletion. MEK inhibition: Inhibitor treatment with 1 μM MEK inhibitor (PD0325901) was performed in 80% confluent cultures for 4 and 24 h respectively. DMSO was used as mock control. Cells were cross-linked (1% formaldehyde, 10 minutes), quenched with 125 mM glycine, washed twice with PBS and harvested in SDS buffer (50 mM Tris at pH 8.1, 0.5 % SDS, 100 mM NaCl, 5 mM EDTA). Cells were pelleted, resuspended in Triton-X IP buffer (100 mM Tris at pH 8.6, 0.3% SDS, 1.7% Triton X-100, and 5 mM EDTA) and chromatin was sonicated (fragment size 200-500bp). 25 µg chromatin (measured by Bradford) was pre-cleared with protein-A Sepharose beads (GE healthcare) for 1 hour and incubated with primary antibody overnight at 4oC. Protein-A Sepharose beads were blocked with 10 µg/ml BSA overnight at 4°C. Next day, beads and antibody/chromatin-mixture were incubated for 3 hours at 4oC. Beads were washed 3x with low salt buffer (1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, pH 8.0, 20 mM Tris-HCl, pH 8.0) and twice with high salt buffer (1% Triton X-100, 0.1% SDS, 500mM NaCl, 2 mM EDTA, pH8.0, 20 mM Tris-HCl, pH8.0). DNA was eluted with elution-buffer (1% SDS, 0.1 M sodium bicarbonate) at 65oC overnight and purified using QIAquick PCR Purification Kit (Quiagen). For ChIP-seq 1-2 ng of ChIP DNA was used for library preparation, using the NEBNext Ultra II DNA library prep kit (E7370; NEB).