Targeted genomic enrichment of long DNA fragments flanking a known core sequence in Arabidopsis thaliana
The goal was to evaluate a novel targeted genomic enrichment strategy to capture and enrich flanking genomic regions surrounding conserved short genomic sequences. The experimental design was as follows: Genomic DNA was extracted from wild-type Arabidopsis thaliana Col-0 leaves using Nucleon PhytoPure Genomic DNA Extraction Kits (GE Healthcare). 100 ug genomic DNA (100 ul @ 1 ug/ 1ul), 300 ul Hybridization Buffer P5 (Invitrogen) and 1 pmole LNA-biotinylated capture probe (1 ul @ 1uM) were placed in a 1.7 ml centrifuge tube and boiled for 5 minutes to denature the genomic DNA. The mix was placed in 45C for 30 minutes for hybridization. 20 ul streptavidin beads from the RiboMinus Plant Kit (Invitrogen) were prepared per the manufacturer's protocol. After 30 minutes of hybridization, the hybridization mix was added to the beads and incubated at 45C for 15 minutes with occasional (every 2-3 minutes) gentle mixing by inversion. Beads were captured with a magnetic stand and were washed 3 times each for 2 minutes with 500 ul 0.1X SSC incubated at 45C. Captured DNA was eluted for one minute with 500 ul nanopure water at 90C twice. DNA was mixed with 1/10 volume 3M sodium acetate pH 5.2, 20 ug glycogen (1 ul @ 20 ug/ul) and 3 volumes of 95% ethanol, vortexed for 30 seconds, and then placed at -20C overnight for ethanol precipitation. DNA was centrifuged at maximum speed at 4C for 20 minutes to spin down pellets. Pellets were washed by 75% ethanol and centrifuged at maximum speed at 4C for 5 minutes and air dried at 4C. DNA was then resuspended in a minimal volume (4-8 ul) of nanopure water. DNA was linearly amplified with the illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare) per the manufacturer's protocol, with the only modification that the incubation time was increased from 1.5 hours to 2 hours. The prepared sample was sent to Sequensys (La Jolla, CA), where it was fragmented into ~ 400 bp and paired-end sequenced on an Illumina GAIIx sequencer.