- NEXTFLEX® cfDNA Enrichment Beads
- NEXTFLEX® End-Repair & Adenylation Buffer Mix 2.0
- NEXTFLEX® End-Repair & Adenylation Enzyme Mix 2.0
- NEXTFLEX® Ligase Buffer Mix 2.0
- NEXTFLEX® Ligase Enzyme 2.0
- NEXTFLEX® PCR Master Mix 2.0
- NEXTFLEX® Primer Mix 2.0 (50 μM)
- Nuclease-free Water
- NEXTFLEX® Resuspension Buffer
- NEXTFLEX® Cleanup Beads 2.0 1.5 mL
REQUIRED MATERIALS NOT PROVIDED
- ~1 ng of Cell-Free DNA in up to 32 µL nuclease-free water.
- NEXTFLEX® DNA Barcodes – 6 / 12 / 24 / 48 (Cat # 514101, 514102, 514103, 514104) or NEXTFLEX-96™ DNA Barcodes (Cat # 514106)
- Ethanol 100% (room temperature)
- Ethanol 80% (room temperature)
- 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
- 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar
- Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
- Magnetic Stand -96 (Ambion, Cat # AM10027) / or / similar
- 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
- Nuclease-free barrier pipette tips
Figure 1. Nucleosome enriched libraries
10 ng input of cfDNA was used for the enrichment (or no enrichment) and prepared using the NEXTFLEX® cell-free DNA-seq kit 2.0 standard protocol with the following enrichments.
- A) No enrichment
- B) Mono-nucleosome enrichment
- C) Mono-, di-, tri-nucleosome enrichment
Figure 2. Automated NEXTFLEX® cell-free DNA-seq kit 2.0 libraries
cfDNA was isolated from 5 mL of plasma using the NextPrep-Mag™ cfDNA automated isolation kit on the chemagic™ 360 instrument and cleaned/concentrated to be eluted in 32 µL into the NEXTFLEX® cell-free DNA-seq kit 2.0 using 9 cycles of PCR (without nucleosome enrichment. Libraries were diluted 1:3 and run on the LabChip® GXII Touch™ HT nucleic acid analyzer.
Papers that Cite the Use of the NEXTFLEX® Cell-Free DNA-Seq Kit:
- Xia, S. et al. (2015) Genomic variations in plasma cell free DNA differentiate early stage lung cancers from normal controls. Lung Cancer. doi: 10.1016/j.lungcan.2015.07.002.
The NEXTFLEX® Cell Free DNA-Seq Kit 2.0 contains enough material to prepare 8 or 48 DNA samples for Illumina® compatible sequencing when bundled with the NEXTFLEX® NGS barcodes. The shelf life of all reagents is 6 months when stored properly. The Resuspension Buffer and Nuclease-free Water can be stored at room temperature. The NEXTFLEX® cfDNA Enrichment Beads and Cleanup Beads 2.0 should be stored at 4°C, and all other components should be stored at -20°C.
Adapters for multiplexing
Kit for cfDNA Isolation