GSM6209250: NCI-H295R, β-catenin, baseline; Drosophila melanogaster; Homo sapiens; ChIP-Seq
Cells were harvested for ChIP-seq with Drosophila melanogaster histone spike-in for all epitopes according to Active Motif's Epigenetic Services ChIP Cell Fixation protocol. Briefly, media was supplemented with 1/10 media volume of freshly prepared Formaldehyde Solution (11% formaldehyde, 0.1 M NaCl, 0.5 M EDTA pH 8.0, 1 M HEPES pH 7.9 in nuclease-free water), and plate was agitated for 15 minutes at room temperature. Fixation was stopped with addition of 1/20 volume of Glycine Solution (2.5 M Glycine, MW 75 in nuclease-free water) and incubating at room temperature for 5 minutes. Cells were then scraped, collected into a conical tube, and pelleted at 800xg at 4°C for 10 minutes. Supernatant was aspirated and each tube of cells was re-suspended in 10 mL chilled PBS-Igepal (0.5% Igepal CA-630 in PBS). Cell were pelleted again, supernatant aspirated, and cells resuspended in 10 mL chilled PBS-Igepal supplemented with 100 μL of 100 mM PMSF in ethanol. Cells were pelleted again, supernatant aspirated, snap frozen on dry ice, stored at -80°C, and shipped on dry ice to Active Motif Services (Carlsbad, CA) for chromatin preparation and ChIP-seq. Chromatin was isolated by addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin (pooled from all submitted samples) with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 μg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were immunoprecipitated using the following antibodies: EZH2 (Active Motif, Cat. No. 39901; concentration 8 μL Ab/30 μg chromatin), H3K27me3 (ActiveMotif, Cat. No. 39155; concentration 4 μg Ab/30 μg chromatin), H3K27ac (ActiveMotif, Cat. No. 39133; concentration 4 μg Ab/30 μg chromatin), SF1 (EMD Millipore, Cat. No. 07-618; concentration 5 μg Ab/30 μg chromatin) and β-catenin (Invitrogen, Cat. No. 71-2700; concentration 4 μg Ab/30 μg chromatin). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. Steps were performed on an automated system (Apollo 342, Wafergen Biosystems/Takara). After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end).