Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
CBFA2T3

Cell type

Cell type Class
Blood
Cell type
M-07e
Primary Tissue
Blood
Tissue Diagnosis
Leukemia

Attributes by original data submitter

Sample

Alias
E-MTAB-43671481641089:MO7EKI ETO2 b
Broker name
ArrayExpress
Description
Protocols: The human MO7e and MO7e-KI AMKL cell lines were cultured in MEM-α supplemented with 20% FBS, Penicillin (100U/mL)-Streptomycin (100µg/mL) and 2mM L-Glutamine (Gibco), and 5 ng/ml of human GM-CSF (PeproTech). HEL cells are cultured in RPMI1640 supplemented with 10% FBS, Penicillin (100U/mL)-Streptomycin (100µg/mL), 2mM L-Glutamine (Gibco). AMKL cells obtained directly from immunodeficient animals or after purification by flow cytometry were cultured in RPMI 1640 medium supplemented with 20% FBS, Penicillin (100U/mL)-Streptomycin (100µg/mL) and 2mM L-Glutamine (Gibco), and 10 ng/ml each of human SCF, IL3, IL6, IL11, TPO, 10U/ml EPO and 5 ng/ml of GM-CSF (PeproTech). ChIP protocol was adapted from the MagnaChIP kit protocol (Millipore). Cells (AMKL and MO7e-KI) were fixed with 1% formaldehyde, lysed with Cell Lysis and then Nuclear Lysis buffers respecting concentration of 20.106 cells per mL, and finally sonicated (30-min cycle on Covaris apparatus; KBioscience). Sheared chromatin was immunoprecipitated overnight using the following antibodies: anti-CBFA2T3 (Abcam, ab33072), anti-ERG (Santa Cruz ; SC354), anti-GATA3 (Santa Cruz ; mix 50/50 of SC268X and SC269X), anti-H3K27ac (ActiveMotif, #39133), anti-H3K4me1 (Diagenode ; #C15410037), anti-H3K4me3 (Diagenode, #C15410003), anti-H3K27me3 (Cell Signaling, #9733S) and rabbit IgG (Upstate, #12-370). 1/10 of the sheared chromatin was used as a reference (Input). Immune complex collection was realized with Protein G Sepharose (Sigma-Aldrich; P3296), 1h30 at +4°C. For GFP-ChIP, GFP-Trap agarose beads were used (Chromotek). Rinses were done according to MagnaChIP kit protocol with Low salt, High salt and LiCL immune complex wash buffers. Finally, elution was realized according the IPure Kit protocol (Diagenode, Cat No C03010012) following manufacturer’s instructions. Enriched DNA from ChIP and Input DNA fragments were end-repaired, extended with an 'A' base on the 3′end, ligated with indexed paired-end adaptors (NEXTflex, Bioo Scientific) using the Bravo Platform (Agilent), size-selected after 4 cycles of PCR with AMPure XP beads (Beckman Coulter) and amplified by PCR for 10 cycles more.
ENA checklist
ERC000011
INSDC center alias
INSERM U1170, Equipe Labellisee Ligue Contre le Cancer, Villejuif, France Institut Gustave Roussy, Villejuif, France Universite Paris Diderot, Paris, France
INSDC center name
INSERM U1170, Equipe Labellisee Ligue Contre le Cancer, Villejuif, France Institut Gustave Roussy, Villejuif, France Universite Paris Diderot, Paris, France
INSDC first public
2017-05-22T17:15:20Z
INSDC last update
2016-12-13T15:18:05Z
INSDC status
public
SRA accession
ERS1473558
Sample Name
ERS1473558
Title
MO7EKI ETO2 b
cell_line
MO7e-KI
disease
acute megakaryoblastic leukaemia
organism
Homo sapiens

Sequenced DNA Library

library_name
MO7EKI ETO2 b_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
The human MO7e and MO7e-KI AMKL cell lines were cultured in MEM-α supplemented with 20% FBS, Penicillin (100U/mL)-Streptomycin (100µg/mL) and 2mM L-Glutamine (Gibco), and 5 ng/ml of human GM-CSF (PeproTech). HEL cells are cultured in RPMI1640 supplemented with 10% FBS, Penicillin (100U/mL)-Streptomycin (100µg/mL), 2mM L-Glutamine (Gibco). AMKL cells obtained directly from immunodeficient animals or after purification by flow cytometry were cultured in RPMI 1640 medium supplemented with 20% FBS, Penicillin (100U/mL)-Streptomycin (100µg/mL) and 2mM L-Glutamine (Gibco), and 10 ng/ml each of human SCF, IL3, IL6, IL11, TPO, 10U/ml EPO and 5 ng/ml of GM-CSF (PeproTech). ChIP protocol was adapted from the MagnaChIP kit protocol (Millipore). Cells (AMKL and MO7e-KI) were fixed with 1% formaldehyde, lysed with Cell Lysis and then Nuclear Lysis buffers respecting concentration of 20.106 cells per mL, and finally sonicated (30-min cycle on Covaris apparatus; KBioscience). Sheared chromatin was immunoprecipitated overnight using the following antibodies: anti-CBFA2T3 (Abcam, ab33072), anti-ERG (Santa Cruz ; SC354), anti-GATA3 (Santa Cruz ; mix 50/50 of SC268X and SC269X), anti-H3K27ac (ActiveMotif, #39133), anti-H3K4me1 (Diagenode ; #C15410037), anti-H3K4me3 (Diagenode, #C15410003), anti-H3K27me3 (Cell Signaling, #9733S) and rabbit IgG (Upstate, #12-370). 1/10 of the sheared chromatin was used as a reference (Input). Immune complex collection was realized with Protein G Sepharose (Sigma-Aldrich; P3296), 1h30 at +4°C. For GFP-ChIP, GFP-Trap agarose beads were used (Chromotek). Rinses were done according to MagnaChIP kit protocol with Low salt, High salt and LiCL immune complex wash buffers. Finally, elution was realized according the IPure Kit protocol (Diagenode, Cat No C03010012) following manufacturer’s instructions. Enriched DNA from ChIP and Input DNA fragments were end-repaired, extended with an 'A' base on the 3′end, ligated with indexed paired-end adaptors (NEXTflex, Bioo Scientific) using the Bravo Platform (Agilent), size-selected after 4 cycles of PCR with AMPure XP beads (Beckman Coulter) and amplified by PCR for 10 cycles more.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
98374753
Reads aligned (%)
45.8
Duplicates removed (%)
47.8
Number of peaks
10523 (qval < 1E-05)

hg38

Number of total reads
98374753
Reads aligned (%)
47.8
Duplicates removed (%)
46.0
Number of peaks
10854 (qval < 1E-05)

Base call quality data from DBCLS SRA