Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
RUNX1T1

Cell type

Cell type Class
Blood
Cell type
Kasumi-1
Primary Tissue
Blood
Tissue Diagnosis
Leukemia Acute Myelogenous

Attributes by original data submitter

Sample

source_name
RUNX1-ETO ChIP with inducible version of CEBPA (C/EBPα-ER) treated with vehicle control-ethanol
cell type
Kasumi-1 cells
genotype
inducible version of CEBPA (C/EBPalpha-ER)
treatment
vehicle control-ethanol
antibody
ETO (Santa Cruz, serial no.:SC9737)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Each aliquot of 2x107 cells was resuspended in 600μl of sonication buffer (Tris-HCL pH8 25mM, NaCL 150mM, EDTA 2mM, Triton 100x 1%, SDS 0.25%, Protease inhibitior cocktail (PIC) 1x). 300 μl of nuclei in sonication buffer was placed in each polystyrene tube and sonicated at 75% amplitude, 26 cycles: 30s on and 30s off per cycle (Q800, Active Motif, USA). Subsequently, 1.2ml of dilution buffer (Tris-HCL pH8 25mM, NaCL 150mM, EDTA 2mM, Triton 100x 1%, glycerol 7.5%, PIC 1x) was added to the pooled post sonication material. This was divided equally between four immunoprecipitations (with 5% of input taken for validation).15 μl protein G beads (Diagenode, Belgium) were washed twice with 500 μl of 50mM citrate phosphate buffer and once with 100mM sodium phosphate). 2μg antibody (EVI-1, C50E12, Cell signalling, lot 3; or RUNX1, Ab23980, Abcam lot 144722) or 4µg antibody (C/EBPα, A2814 Santa Cruz) was added to 10 μl 100mM sodium phosphate, 0.5% BSA and incubated with protein G beads at 4°C for 1 hour. Chromatin was then added to the protein G beads with antibody and returned to 4°C for 4 hours. Unbound chromatin was separated from the beads by magnet and the attached beads were washed by buffer 1 (Tris HCL 20mM, NaCl 150mM, EDTA 2mM, Triton x100 1%, SDS 0.1%), twice with buffer 2 (Tris HCL 20mM, NaCl 500mM, EDTA 2mM, Triton x100 1%, SDS 0.1%), LiCL buffer (Tris HCL 10mM, LiCl 250mM, EDTA 1mM, NP40 0.5%, sodium deoxychlolate 0.5%) and finally twice with wash buffer 4 (Tris HCL pH8, 10mM, NaCl 50mM, EDTA 1mM). The column was eluted twice with 50 μl buffer (NaHCO3 100mM and SDS 1%) and the eluant containing the chromatin was pooled. Crosslinks were reversed by incubating the samples at 65°C overnight in 500mM NaCl, 500 μg/ml proteinase K. DNA was purified by Ampure beads (Beckman Coulter, USA), as above, with the DNA eluted with 50 μl water. Validation of the ChIP was performed by qPCR using a standard curve of genomic DNA from untreated SKH-1 cells (10ng/ μl followed by serial 1:5 dilutions). The input material was diluted 1:5 with water and qPCR was performed as above with primers listed in SI. Validation was analysed as a ratio of the qPCR signal from the ChIP material over the input. Libraries for high throughput sequencing were prepared using the Tru-seq DNA sample preparation kit (Illumina, USA) or Kapa HyperPrep kit (Kapa biosystems, USA), as per manufacturer's protocol. 18 cycles of PCR was performed and 200-350bp fragments were size selected by running the samples in an agarose gel. Libraries were purified from the gel using a Minieluta Gel extraction kit (Qiagen, USA). Libraries were validated by qPCR, with an analysis of the ChIP signal of a positive control region (e.g. PU.1 3H enhancer) over a negative control region (e.g. IVL). Finally, libraries were quantified by Kapa library quantification kit (Kapa biosystems, USA) and run in a pool of four indexed libraries in one lane of a HiSeq 2500 (Illumina, USA) or 12 indexed libraries in one lane of a NextSeq 500 (Illumina, USA) using 50 cycle single-end reads.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
39065319
Reads aligned (%)
44.0
Duplicates removed (%)
43.7
Number of peaks
10105 (qval < 1E-05)

hg38

Number of total reads
39065319
Reads aligned (%)
45.1
Duplicates removed (%)
42.7
Number of peaks
10070 (qval < 1E-05)

Base call quality data from DBCLS SRA