AP28 tissue DNA
Appropriate material from diseased arteries taken at sites of macroscopic atherothrombotic plaque was surgically dissected. Cross-sectional pieces of tissue around 3-5 mm thick were prepared and immediately stored at -80 °C.  DNA was extracted using Qiagen's DNeasy Blood & Tissue kit, following the “Purification of Total DNA from Animal Tissues (Spin-Column)” protocol. Briefly, tissue was cut into tiny pieces of no more than 25mg on a clean petri dish on dry ice, then homogenized in ATL buffer with a 5 mm stainless steel bead (Qiagen) on a Tissuelyser at 50 HZ for 2 min. or until tissue was completely homogenized. After homogenization, proteinase K was added and the sample was incubated at 56°C until it was completely lysed. The remaining steps of the protocol were performed according to Qiagen's recommendation. DNA was eluted with 100uL of Buffer AE.    Prior to library preparation, the quality of the DNA samples was assessed on a Bioanalyzer 2100, using a DNA 12000 Chip (Agilent). Sample quantitation was performed using Invitrogen's Picogreen assay.    Next-generation sequencing library preparation was performed by following Illumina's TruSeq DNA Sample Preparation protocol. The samples were sheared on a Covaris S220 to ~500bp, following the manufacturer's recommendation. Size selection was performed on a Sage Science Pippin Prep instrument, using a 1.5% EtBr agarose cassette and selecting for a tight peak around 620bp. Each library was uniquely tagged with one of Illumina's TruSeq LT DNA barcodes to allow library pooling for sequencing.     Library quantitation was performed using Invitrogen's Picogreen assay and the average library size was determined by running the libraries on a Bioanalyzer DNA 7500 chip (Agilent). Library concentrations were normalized to 4nM and validated by qPCR on a StepOne Plus real-time thermocycler (Applied Biosystems), using qPCR primers recommended in Illumina's qPCR protocol, and Illumina's PhiX control library as standard. Libraries were then pooled and sequenced in two lanes of an Illumina HiSeq2000 sequencing run at a read-length of 101bp paired-end.