GSM5351991: ncd8_PD_S34; Homo sapiens; OTHER
Cells were collected 72h after transfection and lysed in 150 μl prepared DNA lysis buffer (10 mM Tris-HCl, pH 7.5; 0.05% SDS; 25μg/ml proteinase K). DNA lysates were incubated at 37 °C for 1h, followed by an 80 °C enzyme inactivation step for 30 min. For each sample, the target regions were amplified by first round of PCR (PCR1) using gene-specific primers flanking the target sequence. The PCR1 was performed in a 25 μl volume including 100ng genomic DNA, 1.25 μl of 10 μM each primer, 12.5 μl 2 × Phusion High-Fidelity PCR mix (Thermo) and 0.75 μl DMSO. PCR1 reactions were carried out as follows: 98 °C for 2 min, then 34 cycles of (98 °C for 10 s, 65°C for 30 s, 72 °C for 30 s), and a final 72 °C extension for 5 min. PCR1 products were purified by electrophoresis in a 1.5% agarose gel or using AMPure beads. In a secondary 'barcoding' PCR (PCR2), the amplicons were indexed with primer pairs containing appropriate Illumina forward and reverse adaptor sequences. The 25 μl PCR2 was performed with 10ng purified PCR1 products, 2.5 μl of 10 μM forward and reverse barcoding primers, 12.5 μl 2 × Phusion High-Fidelity PCR mix (Thermo) and 0.75 μl DMSO. The PCR2 reactions were carried out as follows: 98 °C for 2 min, then 10 cycles of (98 °C for 10 s, 65 °C for 20 s, and 72 °C for 30 s), followed by a final 72 °C extension for 2 min. PCR2 products were purified by electrophoresis with a 1.5% agarose gel and DNA concentration was measured by fluorometric quantification (Qubit, Thermo). Amplicon libraries were sequenced on the Illumina MiSeq instrument according to the manufacturer's protocols. After demultiplexing, FASTQ files were analyzed using CRISPResso2 with --discard_indel_reads.