Instrument: Illumina HiSeq 3000
Strategy: MNase-Seq
Source: GENOMIC
Selection: MNase
Layout: PAIRED
Construction protocol: To analyze nucleosome occupancy quantitatively in control and TFDP1 knockout cells, We included Drosophila S2 cells as a spike-in control. After mixing 1x106 eHAP cells and 5x104 S2 cells, cells were washed with PBS containing 0.1% BSA and subsequently permeabilized with 0.5% IGEPAL CA-630 in RSB buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 1 mM CaCl2, 3 mM MgCl2 and 0.5 mM PMSF) for 20 min on ice. Nuclei were digested in RSB containing 2.5 U MNase (Takara) at 37 ˚C for 10 min. The reaction was terminated by adding EDTA, EGTA and Proteinase K. The extracted DNA was fractionated by agarose gel electrophoresis, and DNA fragments between 125 and 175 bp representing mono-nucleosome was purified with Monarch PCR & DNA Cleanup Kit followed by treatment with RNase. Libraries for sequencing were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and sequenced on Illumina HiSeq3000 for 100 bp paired-end reads. Two biological replicates were analyzed for each experimental condition.