Driver mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in Acute Myeloid Leukemia
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Blood
Cell type
OCI-AML3
Primary Tissue
Blood
Tissue Diagnosis
Leukemia Acute Myelogenous
Attributes by original data submitter
Sample
ENA first public
2014-12-12
ENA last update
2018-03-08
External Id
SAMEA2043668
INSDC center alias
University of Cambridge
INSDC center name
University of Cambridge
INSDC first public
2014-12-12T09:23:58Z
INSDC last update
2018-03-08T16:11:31Z
INSDC status
public
Submitter Id
E-MTAB-1443:BRD4_IBET
age
2 month
bio source type
frozen_sample
biosourceprovider
ATCC
broker name
ArrayExpress
cell line
OCIAML3
cell type
myelomonocyte
common name
human
disease
acute myeloid leukemia
individual
ATCC_OCIAML3
organism part
blood
phenotype
NPM1c AML
sample name
E-MTAB-1443:BRD4_IBET
sex
male
Sequenced DNA Library
library_name
BRD4_IBET
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Immunoprecipitated DNA was amplified for sequencing using TaqMan PCR mastermix according to the manufacturers instructions. ChIP libraries used for massive parallel sequencing were prepared using the Illumina ChIP-seq kit with the following adjustments. Following chromatin immunoprecipitation and DNA purification, 10-50ng of DNA was used to perform end repair of the ChIP DNA. The following reaction mix was used, DNA (10-50ng resuspended in 30 ml), DNase and RNase free ddH2O 10 ml, T4 DNA ligase buffer with 10 mM ATP 5 ml, 10 mM dNTP mix 2 ml, T4 DNA polymerase 1 ml, Klenow DNA polymerase 1 ml and T4 PNK 1 ml. The sample was incubated for 30 min at 20 C and then purified using the Zymo DNA Clean & Concentrator-5O kit with the DNA eluted in 11 ml of ddH2O. Following end repair adenine bases were added to the 30 end of the DNA fragments using DNA from above 11 ml, ddH2O 33 ml, Klenow buffer 5 ml, dATP 10 ml and Klenow (30 to 50 exonuclease deficient) 1 ml. The sample was incubated for 30 min at 37 C and the DNA was purified using the Zymo kit. Commercially supplied adapters (Illumina; the sequence is commercially protected) were then ligated to the DNA fragments using DNA ligase buffer 14 ml, adaptor oligonucleotide mix 2 ml, DNA ligase 4 ml and DNA 10 ml. The reaction was incubated for 15 min at room temperature and DNA purified using the Zymo kit. Following the ligation of adapters the DNA was amplified by PCR. The reaction conditions included DNA 10 ml, ddH2O 26 ml, 5X Phusion buffer 10 ml, 10 mM dNTP mix 1.5 ml, Phusion polymerase 0.5 ml and 1 ml of both forward and reverse PCR primers the sequence of which is commercially protected. The reaction conditions are as follows: 98 C, 30 s; 98 C, 10 s; 65 C, 30 s; 72 C, 30 s; back to 2 (18 cycles); 72 C for 5 min. Following PCR amplification, the DNA was purified using the MinElute PCR purification kit (QIAGEN). The clean DNA was then size fractionated in a 1.5% agarose gel. Each sample was run in isolation in the gel in a dedicated clean agarose tank in 1 X TAE buffer. The gel was subsequently stained with SybrGreen in a clean staining tank for 1 hr. Following this, 200 -400 bp fragments were excised from the DNA smear present on the gel, and purified using the MinElute QIAGEN Gel-Purification Kit. The completed library was then tested for both size selection and quantity of DNA using a 2100 Agilent Bioanalyser.