GSM8751603: HS/AAV6 - Sample7; Homo sapiens; OTHER
QIAamp DNA Micro Kit (QIAGEN) Custom library construction protocol. PCR starting from >50–100 ng of purified gDNA. The first PCR step was performed with GoTaq G2 DNA Polymerase (Promega) according to the manufacturer's instructions using the following amplification protocol: 95 °C for 5 min, (95 °C for 0.5 min, 60 °C for 0.5 min, 72 °C for 0.5 min) × 20 cycles, 72 °C for 5 min. Forward primer was designed to bind the donor template upstream of the BAR sequence, while the reverse primer annealed outside the homology arm, thus amplifying 328 bp of the on-target integrated cassette. For targeted deep sequencing of the plasmid and AAV libraries, the reverse primer was annealed to the homology arm. The second PCR step was performed with GoTaq G2 DNA Polymerase (Promega) according to the manufacturer's instructions using 5 μl of the first-step PCR product and the following amplification protocol: 95 °C for 5 min, (95 °C for 0.5 min,60 °C for 0.5 min, 72 °C for 0.5 min) × 20 cycles, 72 °C for 5 min. Second-step PCR primers were endowed with tails containing P5/P7 sequences, i5/i7 1000 Illumina tags to allow multiplexed sequencing, and R1/R2 primer binding sites.