HiSeq X Ten paired end sequencing; Circulating Tumor Cell(CTC) Isolation and Genonic varation Detection from Liquid Samples of Lung Cancer
Circulating Tumor Cell(CTC) Isolation and Genonic varation Detection from Liquid Samples of Lung Cancer
Pleural effusion and peripheral blood samples were obtained from lung cancer patients in Shanghai Chest Hospital and Shanghai Municipal Hospital of Traditional Chinese Medicine with written informed consent. The clinical sample collection and experiment were carried out in accordance with guidelines and protocols that approved by the Ethics and Scientific Committees of both hospitals. Single candidate tumor cell whole genome amplification was conducted according to REPLI-g kit (Qiagen) protocol. In briefly, retrieved single cell into 4ul PBS by micromanipulation and added 3ul pre-prepared lysis buffer. Incubated for 10min at 65℃, neutralised reaction with adding 3ul stop solution. Added 29ul amplification buffer, 9ul deionized water and 1ul Phi29 DNA polymerase. Incubation at 30℃ for 8h and inactivation enzyme at 65℃ for 3 min. Amplified genomic DNA was then purified by Gel Extraction Kit (Qigen), and its concentration was determined by Nanodrop. 1ug of DNA amplifed from single cell random fragmented by dsDNA fragmentase (NEB). 150-500bp fragments were purified with Gel Extraction Kit (Qiagen). Applied NEBNext® Ultra End Repair/dA-Tailing Module kit to repair fragments ends and ligated to truncated PE P7 adaptors and barcoded P5 adaptors using NEB Next® Ultra Ligation Module kit. Purified products with Agencourt AMPure XP SPRI beads (Beckman Coulter), enriched the DNA fragments with adaptors by PCR. The coding exons captured with SureSelect All Exon V5 (Agilent Technologies) and posthybridization amplification following the manufacturer’s protocol. The whole exome sequencing was conducted on the Illumina HiSeqTM X Ten platform
Experimental Factor: pleura
organism part