GSM4629347: 293T-sgRNADel_rep1.VEGFA-Detect-Extend-16; Homo sapiens; OTHER
Genomic DNA was freshly extracted using CWBIO universal genomic DNA kit (CWbiotech, CW2298M) and stored in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at -80 °C. Regions flanking the targeting sites were selected for the design of primers, whose overhangs contained the paired Illumina adaptor sequences. In addition, a 10-nt barcode was also added into each primer pairs to lower the detection limit from 10-1 to 10-5 (Primers are listed in the Supplementary). The first round of PCR amplification was performed with NEBNext® Q5® Hot Start HiFi PCR Master Mix (NEB, M0543L) using ~10 ng of genomic DNA as an input template. After ~10 cycles of amplification, the PCR products were purified with 1 × Agencourt AMPure XP beads and eluted with ddH2O. Purified DNA samples were then subjected to the second round of amplification for ~15 cycles and assigned with different indexes followed by a purification with 0.8 × AMPure XP beads. The libraries were quantified with Qubit 2.0 Fluorometer (INVITROGEN) and pooled together for high-throughput sequencing by Illumina HiSeq X Ten (Genetron Health, Beijing).    All FASTQ files in this dataset, have already been grouped by barcode. Meanwhile, the Illumina sequencing errors were trying to be corrected as well (decrease error rate from ~0.1% to ~0.005%). The Python scripts used in this step are available by request to correspondents.
GEO Accession
GSM4629347