NextSeq 500 sequencing; ChIP-seq of neuroblastoma IMR-32, CLB-GA, NGP, IMR-5/75, GI-M-EN, CHP-212, and IMR-5 cell lines to identify TBX2, MYCN, H3K27ac, H3K4me1 and H3K4me3 binding sites
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Neural
Cell type
IMR-32
Primary Tissue
Brain
Tissue Diagnosis
Neuroblastoma
Attributes by original data submitter
Sample
ENA first public
2018-08-16
ENA last update
2018-03-05
ENA-CHECKLIST
ERC000011
External Id
SAMEA104665828
INSDC center alias
Center for Medical Genetics Ghent Cancer Research Institute Ghent
INSDC center name
Center for Medical Genetics Ghent Cancer Research Institute Ghent
INSDC first public
2018-08-16T17:02:55Z
INSDC last update
2018-03-05T10:21:36Z
INSDC status
public
Submitter Id
E-MTAB-6570:Sample 6
broker name
ArrayExpress
cell line
IMR-32
cell type
neuroblast
common name
human
disease
neuroblastoma
individual
IMR-32 MYCN Rep2
metastatic site
abdominal mass
organism part
brain
sample name
E-MTAB-6570:Sample 6
Sequenced DNA Library
library_name
Sample 6_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
A total of 3-5x10^7 cells were crosslinked in complete medium with 1% formaldehyde while shaking for 7 min at room temperature. Crosslinking was quenched with 125 nM glycine, cells were washed twice with PBS and stored at -80°C. Cells were lysed and sonicated with the M220 Covaris for 15 min to obtain 200-300 bp long fragments. Chromatin fragments were immunoprecipitated overnight using 1 ug antibody for every 10^7 cells. 20 ul Protein A UltraLink Resin beads was added per 10^7 cells. Beads were eluted by incubation for 22 min at 5°C while vortexing every 2 min, reverse crosslinking was done at 65°C for 15h. Antibodies used in this experiment are as follows: Goat Anti-TBX2 polyclonal antibody, Santa Cruz Biotechnology, Cat# sc-17880; RRID: AB_2200389; Mouse Anti-N-MYC monoclonal antibody, Santa Cruz Biotechnology, Cat# sc-53993; RRID:AB_831602; Rabbit Anti-H3K4me1 polyclonal antibody, Abcam Cat# ab8895, RRID:AB_306847; Rabbit Anti-H3K4me3 polyclonal antibody; Abcam; Cat# ab8580, RRID:AB_306649; Rabbit Anti-H3K27ac polyclonal antibody, Abcam Cat# ab4729, RRID:AB_2118291 Cells were grown in RPMI1640 medium supplemented with 10% foetal bovine serum, 2mM L-Glutamine and 100 IU/ml penicillin/streptaviding at 37°C in a 5% CO2 humid atmosphere. The chromatin for DNA purposes was resuspended in TE-buffer to dilute SDS in the elution buffer, incubated for 2h at 37°C with 0.2 mg/ml RNase and followed by an incubation of 2h at 55°C with 0.2 mg/ml proteinase K. DNA was isolated using 400 ul phenol:chloroform:isoamylalcohol in phase lockd gel tubes. Upon centrifugation, the aqueous layer was transferred to a new tube with 200 mM NaCl, 30 ug glycogen and 800 ul 100% ethanol, and incubated for 30 min at -20°C. Upon centrifugation, the pellet was washed with 80% Ethanol and resuspended in RNase/DNase free water. Concentration was measured using the Qubit dsDNA HS Assay Kit. Library prep was done using the NEBNext Ultra DNA library Prep Kit from Illumina (E7370S) with 50-500 ng starting material and using 8-12 PCR cycles according to the manufacturer's instructions. Libraries were evaluated on the bio-analyzer (Agilent) using the Agilent high-sensitivity kit, followed by Pippin Prep with a 2% Dye Free Marker L Agarose Gel Cassette to remove large fragments or adapter dimers. Library concentrations were measured with the illumina Kapa Library quantification kit.