Sample information curated by ChIP-Atlas

Antigen

Antigen Class
DNase-seq
Antigen
DNase-Seq

Cell type

Cell type Class
Blood
Cell type
SKH1
NA
NA

Attributes by original data submitter

Sample

source_name
untreated SKH-1 cells
cell type
SKH-1 cells
genotype
untreated cells

Sequenced DNA Library

library_strategy
DNase-Hypersensitivity
library_source
GENOMIC
library_selection
DNase
library_construction_protocol
Prior to DNaseI digestion, apoptotic cells were removed using the Dead Cell Removal Kit (Miltenyl Biotech, UK) as per manufacturer's instructions. 3x 107 SKH-1 cells were suspended in 1ml DNase I buffer (0.3M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 10 mM Tris pH7.4). Digestion on 4.5x106 cells was performed with DNase I (Worthington, DPPF grade) at 80 units/ml in DNase I buffer with 0.4% NP-40 and 2mM CaCl2 at 22ºC for 3 minutes. The reaction was stopped with cell lysis buffer (0.3M NaAcetate, 10mM EDTA pH 7.4, 1% SDS) with 1mg/ml Proteinase K and incubated at 45ºC overnight. For DHS mapping in CD34+ purified t(3;21) patient cells and in SKH-1 transfected with siRNA, lower cell numbers were available and therefore the DNaseI concentrations were reduced according to the cell numbers available.The digested DNAseI material was treated with RNAseA (Sigma Aldrich, Germany) at a final concentration of 100µg/ml at 37ºC for 1 hr. Genomic DNA was extracted using phenol/chloroform method: an equal volume of phenol was added to the reaction and placed on a rotator wheel for 45 minutes. This was centrifuged for 5 minutes at 16000 x g at room temperature. The top layer was transferred to a new tube and the process was repeated sequentially with phenol/chloroform and chloroform. After purification by chloroform extraction, genomic DNA was precipitated with ethanol. This was pelleted by centrifugation for 5 minutes, at 16000 x g at 4°C. The pellet was resuspended with 70% ethanol and centrifugation for 5 minutes, at 16000 x g at 4°C. The pellet was air-dried and dissolved by Tris-EDTA (40mM Tris Acetate 1mM EDTA).Digestion was checked visually by running the samples on a 0.7% agarose gel and by RT-PCR evaluating the ratio of open (TBP promoter) to closed regions of DNA (chromosome 18) and active gene body (beta-actin) to prevent selection of over digested samples (primers in Table S5B). Subsequently, between 2 to 10µg of DNaseI digested DNA (depending on material available) were run on a 1.2% agarose gel for selection of shorter fragments to increase the fraction of fragments captured from DHSs. Prior to loading on gel, the purified DNA was treated again with RNAseA (Sigma Aldrich, USA) at a final concentration of 100µg/ml at 37ºC for 1 hr. 50-300bp fragments were isolated and purified from the gel using a MinElute gel extraction kit (Qiagen, USA) as per manufacturer's instructions and validated by qPCR. Following this, the size selected sample was validated again by RT-PCR, this time using shorter amplicons to enable detection of the shorter fragments enriched by the size selection process. After size selection, a library was prepared using Tru-seq DNA sample preparation kit (Illumina, USA) or MicroPlex library preparation kit v2 (Diagenode, Belgium) as per manufacturer's protocol. After PCR a final size selection step was performed by running the library on 1.5% TAE gel, followed by excision of 190-250bp sized gel fragment. The library was purified from the gel using a MinElute gel extraction kit (Qiagen, USA). The quality of the libraries was assessed on an Agilent 2100 bioanalyser. Libraries were subsequently run on two lanes of an Illumina HiSeq 2500 flow-cell for transcription factor footprinting, or as part of 12 indexed libraries in one lane of a NextSeq500 (Illumina, USA) for DHS mapping alone.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg38

Number of total reads
393836001
Reads aligned (%)
88.5
Duplicates removed (%)
56.4
Number of peaks
55240 (qval < 1E-05)

hg19

Number of total reads
393836001
Reads aligned (%)
87.9
Duplicates removed (%)
58.0
Number of peaks
54167 (qval < 1E-05)

Base call quality data from DBCLS SRA