GSM1513833: H3K4me3 MV4-11R ChIPSeq; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K4me3
Cell type
Cell type Class
Blood
Cell type
MV-4-11
Primary Tissue
Blood
Tissue Diagnosis
Leukemia Acute Myelogenous
Attributes by original data submitter
Sample
source_name
MV4-11R
cell line
MV4-11R
cell type
Acute Myeloid Leukemia (AML) cell line
drug resistance
resistant
ezh2 expression
low
chip antibody
H3K4me3
chip antibody vendor
Cell signaling
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP protocol: Cells were fixed for 10 min in culture media containing 1% formaldehyde and were processed for ChIP as previously described (Dietrich et al, PLOS Genetics 2012). Briefly, formaldehyde fixed chromatin was sonicated in IP buffer (IP buffer =2 volume SDS Buffer : 1 volume Triton Dilution Buffer; SDS Buffer- 100mM NaCl 50mM Tris-Cl, pH8.1, 5mM EDTA, pH 8.0, 0.2% NaN3, 2% SDS; Triton Dilution Buffer- 100mM Tris-Cl, pH 8.6, 100mM NaCl, 5mM EDTA, pH 8.0, 0.2% NaN3, 5.0% Triton X-100) using Branson Sonifier (4 cycles of 30 sec each at 20% amplitude). 15ug DNA (sonicated chromatin) was used for each ChIP in IP buffer. Antibodies used: Rabbit IgG (DAKO), rabbit anti-REST (Millipore, 07-579), anti-SUZ12 (Rabbit monoclonal, Cell signaling). Samples were incubated with antibodies overnight at 40C and immunocomplexes were precipitated with protein A-sepharose beads with 3 hr rotation at 40C. After subsequent washes the samples were decrosslinked overnight at 650C (shaking) in 1%SDS, 0.1M NaHCO3. Finally, ChIP DNA was purified using Qiagen PCR purification kit. The ChIPs were validated at REST and Polycomb target genes by qPCR. ChIP DNA from three parallel ChIPs were pooled and 10 ng was used for making ChIP-seq libraries. The libraries were prepared using “ChIP seq DNA sample preparation kit” from Illumina following manufacturer’s instructions. Individual samples were run in a single lane on HiSeq2000 (Illumina). Basecalls were performed using on-instrument real time analysis (RTA). Mapping was performed with bowtie allowing for <=2 mismatches and only including reads that were mapped to a single position to hg18. The number of reads in 100 bp sections of the genome were counted genome-wide. A normal distribution was fitted to the counts, and the distribution with the least root-mean-square deviation was used as model for the background in the datasets. Thresholds were set so that windows that scored positive had p-values < 0.001 compared to the background distribution. A ChIP-sequenced IgG-control was used as a negative control and handled in the same way. Only windows scoring positive for REST or Suz12, but not IgG, were included for downstream analysis. Also windows where the positive signal (normalized to dataset size) did not exceed the negative signal (normalized to dataset size) at least threefold were omitted from downstream analysis. Datasets were analyzed this way four times in 25 bp shifts, thereby reducing the number of regions that scored negative due to a division of the reads into neighboring sections. Overlapping positive windows and positive windows within 100bp for REST and 500bp for Suz12 of each other were merged. The position of the border was refined by shifting the window in one bp steps until the window scored positive (without scoring positive in the negative control sample) starting at a distance of one window-size from the regions border. Genomic positions were transferred to hg19 using the UCSC liftover tool (http://genome.ucsc.edu/cgi-bin/hgLiftOver). The libraries were prepared using “ChIP seq DNA sample preparation kit” from NEB following manufacturer’s instructions.