GSM8462828: MRSA_Tys; Homo sapiens; ChIP-Seq
Cells from 4 x D150 were pooled for each condition.Step 1 – Cross-Linking and Cell Harvesting: Cells were cross-linked with 1% formaldehyde for 10 minutes to fix protein-DNA interactions. Cross-linking reaction was stopped by adding glycine and cells were harvested, pelleted by centrifugation, and washed with cold PBS.  Step 2 – Cell Lysis and Chromatin Isolation: The cell pellets were lysed with ChIP lysis buffer and bulk chromatin was isolated by centrifugation Step 3 – Chromatin Shearing: Isolated chromatin was sheared to 150-300 bp using a cominbination of enzymatic digestion and sonication.  Step 4 – Chromatin Pre-Clearing and Antibody Incubation: Sheared chromatin was pre-cleared by incubating with Protein A/G agarose beads to reduce non-specific binding. Pre-cleared chromatin was then incubated with acetylated Histone H3K9 antibody overnight.  Step 5 – Immunoprecipitation and Washing: Protein A/G magnetic beads were added to the antibody-bound chromatin fractions to facilitate immunoprecipitation. This incubation was carried out for 2 hours. The antibody-bound chromatin was isolated from bulk chromatin with a magnetic separator. To reduce background signal,  the isolated chromatin was washed with a series of buffers containing progressively higher concentrations of salt and detergent.  Step 6 – Reverse Cross-Linking and DNA Purification: Samples were treated with RNAse to degrade RNA, Proteinase K to degrade proteins, and heat to reverse the cross-linking between proteins and DNA.  Step 7 – DNA Isolation and Storage: DNA was isolated and purified with the Qiagen QIAquick PCR Purification Kit and purified DNA samples were stored at -80°C until ready for sequencing. Libraries were prepered by University of Chicago Core facility using Immumina Library preparation kit and sequenced on the Illumina HiSeq 4000.