Faire-Seq_Scheel_NDGenomics- 4
Mosquitoes were maintained in an insectary at 26°C, ~80% humidity, under a 12 hr light and 12 hr dark cycle with 1 hr crepuscular periods at the beginning and end of each light cycle. Mosquito larvae were fed a suspension of dried beef liver powder, while adults were provided cotton soaked with 10% sugar solution. An artificial membrane blood feeding system was utilized to deliver a blood meal to females.  Females deposited eggs on paper toweling during two hour eggs collections.  Eggs were maintained in the insectary for an additional 49 hrs.      Nuclei were pelleted, resuspended in FAIRE lysis buffer FAIRE Lysis Buffer (2% Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris-Cl pH 8, 1mM EDTA) and subjected to bead beating with 0.5 mm glass beads.  Chromatin was sonicated using a Branson 250 ultrasonifier outfitted with a microtip to an average fragment size of 300-500 bp and then phenol-chloroform extracted.  900 ng of FAIRE-enriched DNA was prepared at the University of Notre Dame Genomics and Bioinformatics Core Facility for sequencing on an Illumina HiSeq 2000 sequencer at the BGI Americas Corporation (Cambridge, MA).    FAIRE DNA Illumina libraries were prepared by the University of Notre Dame Genomics and Bioinformatics Core facility using the TruSeq kit (Illumina) per the manufacturer’s guidelines.  900 ng of FAIRE-enriched DNA was processed following the Illumina TruSeq DNA LT sample preparation protocol.  Briefly, samples were end repaired and 3’ adenylated.  Illumina adapters were ligated to the template, then cleaned up and size-selected on an agarose gel for 100-300 bp fragments.  A 10-cycle PCR reaction enriched for ligation products containing both Illumina adapters.  Sample concentration was measured on a Qubit fluorometer (Life Technologies, Grand Island, NY) and sample size distribution assayed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA).  Next-generation Illumina Sequencing (HiSeq 50 bp paired-end sequencing) with a Illumina HiSeq 2000 (BGI Americas Corporation, Cambridge, MA) was used to generate ~150 million reads for each of three replicate FAIRE DNA preparations.