title: Illumina Genome Analyzer IIx sequencing; H3K4me3 ChIPseq in chicken liver
design: H3K4me3 ChIPseq in chicken liver
library_construction_protocol: Healthy liver from chicken was perfused with 1xPBS and cut in small pieces. The liver tissue was crosslinked in 1% formaldehyde (v/v) and neutralized by adding 250 mM glycine. Afterwards the cells were homogenized by tissue douncing and washed twice with 1xPBS. Cells were resuspended in 10ml Lysis Buffer 1 (50mM Hepes-KOH [pH 7.5], 140mM 5M NaCl,1nM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100, Proteinase Inhibitor). Rotated at 4oC for 10 min. Centrifuged at 2,000g 5 min at 4oC. After removal of the supernatant the pellet was resuspended in 10ml Lysis Buffer 2 (10mM Tris-HCL [pH 8.0], 200mM NaCL, 1mM EDTA, 1mM EGTA, Proteinase Inhibitor). Rotated at 4oC for 5 min. Centrifuge at 2,000g 5 min at 4oC. After removal of the supernatant the pellet was resuspended in 3ml Lysis Buffer 3 (10mM Tris-HCL [pH 8.0], 100mM NaCL, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, Proteinase Inhibitor). Suspension was sonicated (8-12 cycles of 30 sec on and 60sec off) with a micro tip attached to Misonix 3000 sonicator. Tubes were kept on ice water. 30 l 10% Triton X-100 were added to sonicated lysate. Centrifuge at 20,000g for 10 min at 4oC. Immunoprecipitated DNA was end-repaired, A-tailed, ligated to Illumina sequencing adapters, amplified by 18 cycles of PCR and size selected (200-300 bp).