GSM2412278: LSD1 DMSO vehicle; Homo sapiens; ChIP-Seq
THP1 cells were cultured for 48 hours in the presence of DMSO or OG86 250nM at a density of 3x105/ml. Cells were cross-linked at room temperature using 1% formaldehyde. After 10 minutes the reaction was stopped by incubation for 5 minutes with 0.125M glycine. Cell pellets were washed twice with cold PBS containing protease inhibitors (Complete EDTA-free tablets, Roche, Basel, Switzerland). 100 million cells were used per ChIP, as described in the protocol reported by Lee et al. (2006). Briefly, nuclear lysates were sonicated using a Bioruptor Plus (Diagenode) for 15 min at high, 30 sec ON, 30 sec OFF settings. Immunoprecipiation was performed overnight at 20 rpm and 4°C, with 100ul magnetic beads (Dynabeads (Protein G), Invitrogen, Carlsbad, CA) per 10µg antibody. ChIP-grade antibodies were used as follows: LSD1 (ab17721), GFI1 (ab21061) and MYB (ab45150) (Abcam). After washing six times with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl), chromatin IP-bound fractions were extracted at 65°C for 30min with elution buffer (50mM TrisHCl pH8, 10mM EDTA, 1% SDS) vortexing frequently. RNAseA (1mg/ml) and proteinase K (20mg/ml) were used to eliminate any RNA or protein from the samples. Finally DNA was extracted using phenol:chloroform:isoamyl alcohol extraction and precipitated with ethanol (adding 2 volumes of ice-cold 100% ethanol, glycogen (20µg/µl) and 200mM NaCl) for at least 1 hour at -80°C. Pellets were washed with 70% ethanol and eluted in 50ul 10mM TrisHCl pH8.0. ChIP DNA samples were prepared for sequencing using the Microplex Library Preparation Kit (Diagenode) and 1 ng ChIP DNA. Libraries were size selected with AMPure beads (Beckman Coulter) for 200-800 base pair size range and quantified by Q-PCR using Kapa Library Quantification Kit (Kapa Biosystems).