GSM6938201: HCC827 cells, Osimertinib Resistance, 1; Homo sapiens; OTHER
We extract genomic DNA from the cultured cells following the manufacture's protocol (The QIAamp DNA Mini Kit (cat. nos. 51304 and 51306) ). The exome sequences were efficiently enriched from 0.4 μg genomic DNA using Agilent liquidcapture system (Agilent SureSelect Human All Exon V6) according to the manufacturer's protocol. Firstly, qualified genomic DNA was randomly fragmented to an average size of 180-280bp by Covaris S220 sonicator. Remaining overhangs were converted into blunt ends via exonuclease polymerase activities. Secondly, DNA fragments were end repaired and phosphorylated, followed by A-tailing and ligation at the 3'ends with paired-end adaptors. DNA fragments with ligated adapter molecules on both ends were selectively enriched in a PCR reaction. After PCR reaction, libraries hybridize with liquid phase with biotin labeled probe, then use magnetic beads with streptomycin to capture the exons of genes. Captured libraries were enriched in a PCR reaction to add index tags to prepare for sequencing. Products were purified using AMPure XP system (Beckman Coulter, Beverly, USA), DNA concentration was measured by Qubit®3.0 Flurometer (Invitrogen, USA), libraries were analyzed for size distribution by NGS3K/Caliper and quantified by real-time PCR (3 nM). At last, DNA library were sequenced on Illumina for pairedend 150bp reads.