title: CTRL002 H3K9me3 IP
design: "Cells were cross-linked with 1% HCHO for 12 minutes at room temperature, lysed and chromatin sheared using Branson Digital Sonifier. IP were performed overnight on a wheel at 4o with 2.4 micrograms of H3K9me3 antibody (ab8898, Abcam). The following day, antibody-chromatin immunocomplexes were loaded onto Dynal G magnetic beads (Invitrogen 10004D); the bound complexes were washed once in Low Salt Solution (0,1% SDS, 2mM EDTA, 1% Triton X-100, 20mM Tris pH 8, 150 mM NaCl), once in High Salt Solution (0,1% SDS, 2mM EDTA, 1% Triton X-100, 20mM Tris pH 8, 500 mM NaCl), once again in Low Salt Solution and once in Tris/EDTA 50 mM NaCl. Crosslinking was reversed at 65oC overnight in Elution Buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS) and DNA extracted from beads by standard phenol/chloroform extraction, precipitated and resuspended in 30 ul of 10 mM Tris pH 8. ChIP efficiency was tested by qPCR reactions, performed in triplicate (precipitated DNA samples as well as input DNA) using QuantiTect SYBR Green master mix (Qiagen) on a StepOnePlus Real-Time PCR System (Applied Biosystems). Relative enrichment was calculated as ChIP / Input ratio. Libraries for sequencing were created using the automation instrument Biomek FX (Beckman Coulter), then qualitatively and quantitatively checked using Agilent High Sensitivity DNA Kit (Agilent Technologies, 5067-4627) on a Bioanalyzer 2100 (Agilent Technologies). Libraries with distinct adapter indexes were multiplexed and, after cluster generation on FlowCell, were sequenced for 50bp in the single read mode on a HiSeq 2000 sequencer at the IEO Genomic Unit in Milan."