Instrument: Illumina HiSeq 3000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Cells were fixed using 1% methanol-free formaldehyde (CAT no) in DMEM (HEK293) or IMDM (K562) at room temperature for 10 minutes, followed by 5 minutes blocking in 0.125M glycine. Cells were washed twice with ice-cold PBS. Cell pellet was resuspended in Farnham Buffer (5 mM PIPES pH 8; 85 mM KCl; 0.5% Igepal). Cell suspensions were sonicated in 1ml Covaris tubes using Covaris S220 with the following settings: Peak Power=75; Duty Factor=2; Cycles/Burst=200. Sonication time varied from cell type to cell type; K562=~2.5minutes and HEK293=~3minutes. Isolated nuclei were washed with Farnham Buffer and suspended in shearing buffer (10 mM Tris-HCl pH 8; 0.1% SDS; 1 mM EDTA). Chromatin was sheared by sonication in 1ml Covaris tubes using the following settings: Peak Power=140; Duty Factor=5; Cycles/Burst=200, Time=25-30 minutes. Debris was removed by centrifugation. A DNA fragment-size distribution of 200-600bp was considered as ideal chromatin for ChIP. Chromatin was diluted 1:1 with IP buffer to achieve a final 0.05% SDS concentration. 200 μg of good quality chromatin was used for immunoprecipitation. Protein A or G magnetic beads (CAT) were incubated (rotated) with 5-10μg of antibody for 6h at 4ºC. This bead-antibody complex was then incubated overnight at 4ºC with chromatin.