GSM4294389: 542_BM_C1; Homo sapiens; OTHER
QIAamp DNA Micro Kit (QIAGEN) Custom library construction protocol. PCR amplicons for each individual sample were generated by nested PCR starting from >50-100 ng of purified gDNA. The first PCR step was performed with GoTaq Hot Start Polymerase (Promega) according to manufacturer instruction using the following amplification protocol: 95°C x 5', (95°C x 0.5', 60°C x 0.5', 72°C x 0.5') x 20 cycles, 72°C x 5'. Forward primers were designed to bind donor template upstream the BAR sequence, while the reverse primers annealed outside the homology region, thus amplifying 328 bp of the on-target integrated cassette. Exclusively for targeted sequencing of the transfer vector plasmid and AAV library, the reverse primer annealed to the downstream homology region. The second PCR step was performed with GoTaq G2 DNA Polymerase (Promega) according to manufacturer instruction using 5 µl of the first-step PCR product and the following amplification protocol: 95°C x 5', (95°C x 0.5', 60°C x 0.5', 72°C x 0.5') x 20 cycles, 72°C x 5'. Second-step PCR primers were endowed with tails containing P5/P7 sequence, i5/i7 Illumina tag to allow multiplexed sequencing and R1/R2 primer complementary sequence. PCR library amplicons were separately purified using MinElute PCR Purification kit (QIAGEN) and AmpPure XP beads (Invitrogen). NextTera Illumina
GEO Accession
GSM4294389