ChIP seq on steatotic IHH cells with gamma H2AX ab
IIHH cells were grown in control media (10% FBS (Life Technologies, 10108-165), 1% PEN/STREP (Life Technologies 15140-122), 0.1% L-Glutamine (Life Technologies 25030-024), 0.02% Dexamethasone (Sigma D49025MG), 0.1% double-diluited Insulin Human Recombinant Zinc (Life Technologies 12585-014), and oleic acid supplemented (300uM) media for 4 days.  Chromatin was isolated from 20 million cells using the Magna ChIP A/G Kit (One-day chromatin Immunoprecipitation Kits, Merck) according to manufacturers instructions. Chromatin was sonicated with a Vibra-Cell VCX750 probe sonicator (Sonics) for 3 pulses of 15s separated by 30s intervals at 30% output. 50ul of chromatin was immunoprecipitated with 5ug antibody specific for Anti-gamma H2A.X (phospho S139) antibody - ChIP Grade (Abcam) or with 5ug antibody specific for histone H4k16ac (ActiveMotif). 100-250ng of isolated DNA (average size 500bp) was sent to Genewiz for sequencing in an Illumina HiSeq 2x150bp. Sequencing yield was 29-37Mb per sample. Sequence analysis was also performed by Genewiz. Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality at 3 end (error rate > 0.01) using CLC Genomics Server 9.0. Trimmed data was then aligned to reference genome for human genome hg38. During the mapping, only specific alignment was allowed. 163-198M reads were aligned (all >95%).