title: GSM2309350: Macrophage, intermittent hypoxia H3K9ac; Mus musculus; ChIP-Seq
library_construction_protocol: C57BL/6J were divided in two groups:  chronic intermittent hypoxia (CIH) and room air controls (RA). Mice from CIH groups housed in identical commercially designed chambers (30"x20"x20") that can accommodate 24 mice each, and are operated under 12 hour light-dark cycle (Oxycycler model A44XO, Biospherix). A custom oxygen concentration profile for the 12 daylight hours was originally extrapolated from the saturation recordings of an adult patient with moderate to severe obstructive sleep apnea, which consists of 90 sec of 6.1% O2 balance nitrogen alternating with 90 sec 21% O2 (room air for 12 hours during the light phase. The O2 concentration in the chambers was continuously monitored and automatically adjusted throughout the 12 hours of light time (07:00 a.m.-07:00 p.m.). For the remaining 12 hours of nighttime, oxygen concentrations were kept at 21% O2. Chronic intermittent hypoxia exposures lasted 20 weeks. Mice in the RA group were housed in matching cages and exposed to 21% O2 oscillations. Following exposures, mice were sacrificed using CO2 intoxication and exsanguination via cardiac puncture. Aortas were perfused with 5-10ml of ice-cold PBS supplemented with 20IU/ml heparin, dissected and cleaned under the microscope from surrounding adipose and lymphoid tissues. Dissected aortas were minced and incubated with an aorta dissociation cocktail for 1hr on a shaker at a speed of 35 RPM at 37° C. The cocktail included 450u/ml of Collagenase type I (Worthington Biochemical, Lakewood, NJ, USA), 125 U/ml of Collagenase type XI, 60 U/ml of DNASE I and 60 U/ml of Hyaluronidase (all from Sigma-Aldrich, St. Louis, MO, USA), diluted in PBS + 20mM HEPES. Following incubation, the digestion reaction was stopped with 5% fetal bovine serum in PBS, cells were passed through 100 µm filter, washed and re-suspended for further processing in flow buffer (PBS with 2% FBS). Next, CD11b+ cells were isolated using magnetic beads with an EasySep positive selection kit (STEMCELL Technologies Inc., Vancouver, Canada) per manufacturer's protocol. Average yield per single mouse aorta was 2,000-4,000 cells. Cell pellets were stored at -80°C until processing. Macrophages were isolated from aortas of mice from CIH (n=20) or RA (n=20). Immediately after isolation, cells were crosslinked and chromatin immunoprecipitated using the True MicroChip kit (Diagenode, Denville NJ) and antibodies specific for H3K9ac and H3K27me3 histone modifications, according to manufacturer's instruction. DNA was purified using MicroChip columns (Diagenode), according to manufacturer's instruction. Sequencing libraries from immunoprecipitated aorta macrophage DNA were prepared using the MicroPlex kit (Diagenode) according to the manufacturer's protocol. In brief, immunoprecipitated DNA was quantified using Picogreen staining protocol (Quant-iT™ PicoGreen® dsDNA Assay Kit, ThermoFisher Scientific, Grand Island, NY) and 10 pg were used as input material. DNA was repaired and end-blunted by enzymatic treatment. Stem-loop adaptors with blocked 5' ends were ligated to the 5' end of the genomic DNA, leaving a nick at the 3' end. The 3' ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes were added through amplification. Libraries were purified using AMPure XP beads protocol (Beckman Coulter, Indianapolis, IN) and quantified using the KAPA Library Quantification Kit for Illumina Sequencing Platforms (Kapa Biosystems, Wilmington, MA). Libraries' fragment size distribution was verified using the Bioanalyzer high sensitivity DNA chip (Agilent Technologies, Santa Clara, CA). Libraries were pooled and spike-in PhiX Control v3 (Illumina, San Diego, CA) was added.  Clusters were generated and sequenced using a HiSeq2000 instrument (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM2309350