Tapestation high sensitivity d5000 screentape

The gDNA samples for Illumina sequencing were fragmented using the Covaris to 400 bp following the manufacturer recommended protocol. The genomic libraries were constructed using 100 ng as the input and the NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs). Three PCR cycles were performed with each library prior to library validation using the TapeStation High Sensitivity D5000 ScreenTape (Agilent Technologies). Libraries were quantified using the Qubit 1 × dsDNA HS Assay kit (ThermoFisher Scientific) and molar concentration was calculated to pool the libraries in equimolar ratios. The pool was then quantified and 14 pM was loaded into the MiSeq flow cell. The run was set as a 300 paired-end run using the 600-cycles v3 kit.

cDNA was made from RNA and amplified using the SMART-Seq version 4 Ultra Low Input RNA Kit according to the manufacturer’s protocol (Takara Bio). Undiluted RNA samples from 2,000–8,000 cells were used for cDNA amplification with 14 cycles (Supplemental Table 2). The cDNA was purified by the Agencourt AMPure XP Kit (Beckman Coulter) and the quality assessed by TapeStation (High Sensitivity D5000 ScreenTape kit) according to the manufacturer’s protocols (Agilent Technologies). The samples were stored at –20°C before use.
Library preparation was performed using the Nextera XT DNA Library Preparation kit and the Nextera XT Index kit according to the manufacturer’s protocol (Illumina). The input cDNA was diluted according to the results from the TapeStation (Supplemental Table 2), and unique combinations of i5 (5 μL) and i7 (5 μL) index adapters were added to each respective sample. The libraries were assessed by TapeStation High sensitivity D5000 ScreenTape (Agilent Technologies), consisting of fragments mostly of 250–1,500 bp, with a peak of 600–1,000. Sequencing of the products was performed at the Bergen Genomics core facility, using a HiSeq 4000 sequencer according to the Illumina TruSeq Stranded mRNA protocol.

The QIAamp MinElute ccfDNA Mini Kit (Qiagen, Hilden, Germany) was used to isolate cfDNA from 2 mL plasma according to the manufacturer's instructions, and samples were eluted in 25 μL.
The cfDNA concentration was measured using the Qubit Fluorometric Quantitation System (Thermo Fisher Scientific, Waltham, MA, US). Quality measurements were performed with TapeStation (High Sensitivity D5000 ScreenTape; Agilent Technologies, Santa Clara, CA, US). Statistical testing of differences in ctDNA concentrations was assessed using Wilcoxon signed‐rank test in R.