GSM2043467: Lur_ChemSeq; Homo sapiens; OTHER
A549 cells treated with 300nM of Biotin-Lurbinectedin during 4h were cross-linked with 1% formaldehyde at room temperature for 10 min. After sonication, the soluble chromatin was incubated with 80ul of Streptavidin beads (Life technologies) over-night. After wash the captured DNA was eluted, de-cross-linked and purified by QiAquick Spin columns (Qiagen). The DNA was used to construct a library and sequenced on a Illumina HiSeq 2500 system. ChIP-seq (Chem-seq) ChIP-seq libraries were prepared using NEXTflex ChIP-Seq Kit (#5143-02, Bioo Scientific) following the manufacturer's protocol (V12.10) with some modifications. Briefly, around 10 ng of ChIP enriched DNA were end repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK, then size selected and cleaned-up using Agencourt AMPure XP beads (#A63881,Beckman). A single ‘A’ nucleotide was added to the 3’ ends of the blunt DNA fragments with a Klenow fragment (3' to 5'exo minus). The ends of the DNA fragments were ligated to double stranded barcoded DNA adapters (NEXTflex ChIP-Seq Barcodes - 6, #514120, Bioo Scientific) using T4 DNA Ligase. The ligated products were enriched by PCR (2 min at 98°C; [30 sec at 98°C, 30 sec at 65°C, 60 sec at 72°C] x 14 cycles; 4 min at 72°C) and cleaned-up using Agencourt AMPure XP beads. Prior to analyses DNA libraries were checked for quality and quantified using a 2100 Bioanalyzer (Agilent). The libraries were loaded in the flowcell at 8pM concentration and clusters were generated using the Cbot and sequenced on the Illumina Hiseq2500 as single-end 50 base reads following Illumina’s instructions.
GEO Accession
GSM2043467