Qubit dna dsdna assay kit

Blood samples were collected in cfDNA BCT tubes containing a formaldehyde-free preservative reagent that could stabilize nucleated blood cells.6 (link),7 (link) Plasma was separated by centrifugation (1600 xg for 10 min) and then transferred to 1.5 mL Eppendorf tubes (Axygen, NY, USA) for further centrifugation at 16,000 xg for 10 min at 4°C. Plasma cfDNA was isolated from 1–2 mL plasma with the Mag MAX^TM cell-free DNA isolation kit (Life Technologies, CA, USA). DNA was extracted from white blood cells with the TIANamp blood DNA kit (TIANGEN, Beijing, China). Tumor DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) specimens with the blackPREP FFPE DNA kit (Analytik Jena, Jena, Germany). The concentration of DNA was quantified by the Qubit DNA dsDNA assay kit (Life Technologies, CA, USA).

For cfDNA samples, 10–50 ng DNA was used for library construction. For the blood cell and FFPE DNA samples, 200–500 ng DNA was sheared into 200-bp fragments with a Covaris M220 ultrasonicator (Covaris, MA, USA) according to the recommended protocol. Libraries were prepared with a KAPA Hyper Preparation kit (Kapa Biosystems, MA, USA). According to the DNA quality prior to PCR, the ligated fragments were amplified for 6–11 cycles. DNA was purified by using Agencourt AMPure XP beads (Beckman-Coulter, CA, USA), and double size selection was conducted during library preparation. The Qubit DNA dsDNA assay kit (Life Technologies, CA, USA) was used to quantify the libraries, and a 2100 bioanalyzer with the DNA 1000 kit (Agilent, CA, USA) was used to determine fragment length.

NimbleGen SeqCap Hybridization and Wash kits (Roche NimbleGen, Switzerland) were used according to the technical note for targeted region selection. The hybridized probes can capture 1 µg library DNA from 8–12 indexed Illumina libraries. The probe library was designed using the Nimble Design portal (Version 02) and the genome build kit hg19 NCBI Build 37.1/GRCh37. The captured DNA fragments were divided into two 50-µL reactions (14 PCR cycles). Agencourt AMPure XP beads (Beckman-Coulter, CA, USA) were used to pool and purify these reactions. The Qubit DNA dsDNA assay kit (Life Technologies, CA, USA) was used to quantify the libraries, and then, the captured product was sequenced by using 150-bp paired-end runs on an Illumina HiSeq X10. A 2100 bioanalyzer with the DNA 1000 kit (Agilent, CA, USA) was used to determine fragment length.