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
SAMEA4608317
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_KO_DMSO_IgG
broker name
ArrayExpress
cell line
G144
cell type
glial cell
common name
human
developmental stage
adult
disease
glioblastoma multiforme
genotype
CRISPR/Cas9 mediated knockout of CIC
organism part
brain
sample name
E-MTAB-6682:G144_KO_DMSO_IgG

Sequenced DNA Library

library_name
G144_KO_DMSO_IgG_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).

Sequencing Platform

instrument_model
NextSeq 500

hg38

Number of total reads
12854560
Reads aligned (%)
95.5
Duplicates removed (%)
14.2
Number of peaks
596 (qval < 1E-05)

hg19

Number of total reads
12854560
Reads aligned (%)
94.6
Duplicates removed (%)
15.8
Number of peaks
545 (qval < 1E-05)

Base call quality data from DBCLS SRA