GSM6470188: KM-H2_DHS_1; Homo sapiens; DNase-Hypersensitivity
Dnase-Seq was essentially performed as previously described (Kreher et al 2015) with slight modifications. Briefly, cells were washed and resuspended at 108 cells/mL in ice-cold ψ buffer (11 mM KPO4, pH 7.4, 108 mM KCl, 22 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 1 mM dithiothreitol, 1 mM ATP). 1 million Reh, KM-H2 or L428 cells were treated with 12U/mL DnaseI (Worthington) for 3 mins at 22°C. Digestion was stopped with the addition of 200µL lysis buffer (100 mM Tris, pH 8.0, 40 mM +EDTA, 2% SDS, 200 μg/ml proteinase K) overnight at 37°C. Dnase digestion efficiency was checked via low-voltage overnight electrophoresis (10V) on a 0.5% TAE agarose gel. Short-fragment size-selection was performed by cutting out gel bands between 100-200 bp and subsequent purification using the QiaQuick gel extraction kit (Qiagen) according to manufacturer's instructions For SOLiD libraries, library preparation was performed using the AB SOLiD 4 System library kit (SOLiD Fragment Library Oligos Kit, SOLiD Barcoding Kit Modules 1-96, SOLiD Fragment Library Construction Kit Reagents , Agencourt AMPure XP, 60 mL Kit, SOLiD Library TaqMan Quantitation Kit, Quanti-iT™ dsDNA HS Assay Kits, 500 Assays, 1x Low TE Buffer, 95% ethanol) according to manufacturer's instructions and sequenced on an AB SOLiD 4 System sequencer. For illumina libraries, library preparation was performed using the KAPA hyperprep kit (Roche) following manufacturer's guidelines. Library quality was checked via qPCR using TBP, ACTB and gene desert control oligonucleotides (Bevington et al 2016). Libraries were sequenced at 400 million reads per library in single-end mode on separate lanes using an Illumina HiSeq 2000 system according to manufacturer's instructions.