Antigen

Antigen Class

No description

Antigen

NA

Cell type

Cell type Class

Neural

Cell type

Neural Stem Cells

MeSH Description

Self-renewing cells that generate the main phenotypes of the nervous system in both the embryo and adult. Neural stem cells are precursors to both NEURONS and NEUROGLIA.

Sample

ENA first public

2018-05-30

ENA last update

2018-04-24

ENA-CHECKLIST

ERC000011

External Id

SAMEA4608341

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:hNSC_18_5_24h_CIC

age

18.5

broker name

ArrayExpress

cell type

neural stem cell

common name

human

developmental stage

late embryo

disease

normal

genotype

wild type genotype

organism part

brain

sample name

E-MTAB-6682:hNSC_18_5_24h_CIC

Sequenced DNA Library

library_name

hNSC_18_5_24h_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).

Sequencing Platform

instrument_model

NextSeq 500

hg38

Number of total reads

44176759

Reads aligned (%)

96.5

Duplicates removed (%)

36.1

Number of peaks

1367 (qval < 1E-05)

hg19

Number of total reads

44176759

Reads aligned (%)

95.5

Duplicates removed (%)

37.4

Number of peaks

1109 (qval < 1E-05)