title: Illumina Genome Analyzer IIx sequencing; Pol III ChIP-seq of liver cancer cell lines
design: Pol III ChIP-seq of liver cancer cell lines
library_construction_protocol: Liver hepatocellular carcinomas cell lines HepG2 and Huh7 from human and Hepa1-6 and Hepa-1c1c7 from mouse were grown to 80% confluency in DMEM (Sigma) supplemented with 10% foetal bovine serum (FBS) and antibiotics (100 g/l penicillin and 100 g/ml streptomycin) at 37C and 5% CO2. Cells were fixed in 1% formaldehyde (v/v). After 20 minutes, the cross-linking reaction was quenched with 1/20 volume of 2.5 M glycine to neutralize the formaldehyde. 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 (200300 bp).
attributes.tag: Experimental Factor: human
attributes.value: organism
attributes.tag: Experimental Factor: Huh7
attributes.value: cell line