title: E2846
design: Exome Capture
library_construction_protocol: "Genomic DNA was isolated using the FastDNA kit (MP Biomedicals). Approximately 500ng to 1?g of DNA was used to produce genomic sequencing libraries: DNA was fragmented using dsDNA Fragmentase (New England Biolabs). DNA fragments were purified using AMPure beads (Beckman Coulter). After end repair using End Repair enzyme (New England Biolabs), a deoxyadenosin was added at the 3? end of the fragments using 3? to 5? Exo-?? Klenow fragment (New England Biolabs). Custom synthesized six base barcoded adapters or NEXTflex DNA barcoded adapters (Bioo Scientific) were used. The adapter ligated libraries were size selected for an average insert size of 300bp using AMPure beads.?The pre-capture amplification step was performed using the standard NimbleGen protocol. Equal amounts of library products from?10 to 32 genomic libraries?were pooled to obtain total of 1 ?g DNA for the hybridization.?Exome enrichment was performed using the Roche/NimbleGen Seq-EZ kit: hybridization was performed for 72 hours using a biotinylated custom oligonucleotide library corresponding to selected exons of?Oryza sativa?var Nipponbare (baits). Genomic DNA-bait hybrids were captured using Streptavidin magnetic beads, washed, and amplified by PCR using Post-capture primers (8 cycles).?The final pooled libraries were sequenced according to the manufacturer's recommendations (Illumina, San Diego, CA)."