title: AAV5 infection HeLa Sample B
design: Experiment design: Integrant-Capture Sequencing DNA Oligo Sequences: Sequences of the pLinker primer and asymmetric linker oligos were described previously. The primer sequences utilized for AAV-5 were: P1: 5’-[Bio-TEG]CAGAGCAAAGAATGGCGGCGG-3’ and P2: 5’-GCGGGCTCGTTCACTCGGTC-3’.  HeLa cells were infected with purified and concentrated AAV-5 at 1E4 genomes/cell. Cells were grown for a three weeks post-infection period with passaging as needed into fresh 150cm2 flasks. Five aliquots of 2.5 million HeLa cells, containing ~250ug of genomic DNA in total, were harvested by trypsinization and washed with PBS. Aliquots were lysed in Proteinase K buffer (100 mM Tris (pH 8), 0.2% SDS, 200 mM NaCl, 5 mM EDTA) with 200 ug/mL Proteinase K. Genomic DNA was purified by phenol-chloroform extraction and ethanol precipitation. Sonication was conducted using a Bioruptor (Diagenode) to generate DNA smears of 500-1200bp, with an 850bp core. DNA was polished using the End-It DNA Repair Kit (Epicenter), purified, and dA-tailed utilizing the 3´-5´ exo– Klenow Fragment (NEB). Then, fragments were ligated to 200pmol of annealed linkers. Viral Junction Amplification: All PCRs were conducted using Herculase II Fusion DNA Polymerase (Agilent Technologies) according to manufacturer specifications. Pooled, linker-ligated DNA was divided into 800ng aliquots and subjected to linear-amplification (single-primer) PCR with biotinylated SP-1: 1x(98C-3min) 12x(98C-40s, 65C-30s, 72C-45s) 1x(72C-1min). Reactions were then spiked with pLinker and subjected to exponential PCR amplification: 1x(98C-3min) 35x(98C-40s, 65C-30s, 72C-45s) 1x(72C-5min). Amplification products 400bp-1.2kb were isolated by agarose gel electrophoresis, and virus primer-specific products were enriched by magnetic streptavidin bead pull-down. Semi-nested PCR was performed with SP-2 and pLinker: 1x(98C-3min) 35x(98C-40s, 65C-30s, 72C-40s) 1x(72C-5min). Amplification products 400bp-1.2kb were isolated by agarose gel electrophoresis. Paired-End Library Production and Sequencing: Linkers were digested with AscI and removed by agarose gel purification. Fragments were then polished, purified, and dA-tailed as after the genomic DNA sonication. Fragments were then ligated to Illumina paired-end adapters and isolated by agarose gel electrophoresis. A final, 30-cycle, library PCR was conducted utilizing Illumina primers PE1.0 and PE2.0 according to manufacturer specifications, and amplification products 350bp-1kb were isolated by agarose gel electrophoresis. The final libraries were submitted to 50x50 paired-end deep sequencing using an Illumina HiSeq 2000.
identifiers.namespace: Weill Medical College of Cornell University
identifiers.id: AAV5/HeLa B