Labchip gx touch ht genomic dna reagent kit

Germline DNA was extracted from either 650 ul of saliva collected in an Oragene sample tube or 400 ul of blood collected in a PAXGene blood tube. Oragene and PAXGene tubes were selected because they contain preservatives to stabilize the samples and thus mitigate issues related to transportation time and/or temperature fluctuations. Tumor total nucleic acid was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue sections that were macrodissected (if needed based on Pathologist assessment of tumor cellularity) and proteinase K digested. For all sample types, total nucleic acid was extracted with a Chemagic360 instrument using a source-specific magnetic bead protocol and stored at 4°C if less than 24 hours and −80°C if longer. Total nucleic acid was utilized for all DNA library construction. RNA was purified from the total nucleic acid by DNaseI digestion and magnetic bead purification. Nucleic acids were quantified by a Quant-iT picogreen dsDNA reagent Kit or Quant-iT Ribogreen RNA Kit (Life Technologies), and quality was confirmed using a LabChip GX Touch HT Genomic DNA Reagent Kit or LabChip RNA High HT Pico Sensitivity Reagent Kit (PerkinElmer).

For the PVSRIPO cohort, germline DNA was extracted from blood collected in a PAXGene blood tube. Total nucleic acid was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue sections that were microdissected (if deemed necessary by pathologist prediction of tumor cellularity) and digested with proteinase K. Total nucleic acid was extracted with a Chemagic360 instrument from both blood and tumor samples using a source-specific magnetic bead protocol. Total nucleic acid was the input for all DNA library construction; RNA was purified from total nucleic acid via DNaseI digestion followed by magnetic bead purification. Nucleic acid quantification was performed by a Quant-iT picogreen dsDNA reagent Kit or Quant-iT Ribogreen RNA Kit (Life Technologies). Quality was tested using a LabChip GX Touch HT Genomic DNA Reagent Kit or LabChip RNA High HT Pico Sensitivity Reagent Kit (PerkinElmer). Sequencing (WES and RNAseq) was performed on an illumina Hi-Seq 4000 system using patterned flow cell technology at TEMPUS, Inc.

DNA and RNA sequencing was performed on clinical tumor specimens and saliva samples (from the same patients as the tumor specimens) for 6 of the 8 patients using the Tempus xT assay [26 (link)]. Briefly, nucleic acid was extracted from tumor tissue sections with tumor cellularity greater than 20% using a Chemagic360 instrument and a source-specific magnetic bead protocol. Total nucleic acid was used for DNA library construction, while RNA was further purified by DNase I digestion and magnetic bead purification. The nucleic acid was quantified with a Quant-iT PicoGreen dsDNA Kit or Quant-iT RiboGreen RNA Kit (Life Technologies), and quality was confirmed with a LabChip GX Touch HT Genomic DNA Reagent Kit or LabChip RNA High HT Pico Sensitivity Reagent Kit (PerkinElmer).
For DNA library construction, 100 ng DNA from tumor or normal samples was mechanically sheared to an average size of 200 bp using a Covaris ultrasonicator. The libraries were prepared using the KAPA Hyper Prep Kit. Briefly, DNA underwent enzymatic end repair and A-tailing, followed by adapter ligation, bead-based size selection, and PCR. The captured DNA targets were amplified using the KAPA HiFi HotStart ReadyMix. The amplified target-captured libraries were sequenced on an Illumina HiSeq 4000 System with patterned flow cell technology.