P-GSE48216-6 - P-GSE48216-6
nucleic acid library construction protocol
Qiagen Gentra Puregene Cell Kit Exome libraries of matched pairs of tumour / normal genomic DNAs were generated using the Agilent SureSelect XT kit and Agilent Automation Systems NGS system per manufacturer’s instructions. 1 ug of each genomic DNA was sheared using a Covaris E220 to a peak target size of 150 bp. Fragmented DNA was concentrated using AMPureXP beads (Beckman Coulter), and DNA ends were repaired using T4 DNA polymerase, Klenow polymerase, and T4 polynucleotide kinase. 3’ A-tailing with exo-minus Klenow polymerase was followed by ligation of Agilent paired-end oligo adapters to the genomic DNA fragment. Ligated DNA was PCR amplified for 8 cycles and purified using AMPure XP beads and quantitated using the Quant-It BR kit (Invitrogen). 500 ng of sample libraries were hybridized to the Agilent biotinylated SureSelect 37Mb Capture Library at 65°C for 72 hr following the manuufacturer’s protocol. The targeted exon fragments were captured on Dynabeads MyOne Strepavidin T1 (Invitrogen), washed, eluted, and enriched by amplification with Agilent post-capture primer and an indexed reverse primer for multiplexing12 additional cycles. After purification of the PCR products with AMPure XP beads, the quality and quantity of the resulting exome libraries were analyzed using an Agilent Bioanalyzer High Sensitivity chip.