Qubit high sensitivity dna kit

Patients selected for sequencing had tumor purity above 90%. After extraction, RNA quantity was evaluated using the Qubit RNA Assay Kit (Life Technologies, Carlsbad, CA) and RNA quality was determined on the Bioanalyzer using the RNA Pico Kit (Agilent, Santa Clara, CA). We used 100 ng of total RNA for each sample. Next, library preparation was done with NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA), was converted into a DNA library following the manufacturer’s protocol, with no modifications. Library quantity was determined using the Qubit High Sensitivity DNA Kit and library size was determined using the Bioanalyzer High Sensitivity Chip Kit (Agilent). Finally, libraries were put through quantitative PCR using the Universal Library Quantification Kit for Illumina (Kapa Biosystems, Wilmington, MA) and run on the 7900HT Fast quantitative PCR machine (ABI, Grand Island, NY). Libraries passing QC were diluted to 2 nM using sterile water, and then sequenced on the HiSeq 2000 system (Illumina, San Diego, CA) at a final concentration of 12 pM, following all manufacturer’s protocols. The patients retained in this study had a sequencing depth between 9.4 and 150 million reads. As a sanity check, we verified that the number of fusions identified in each patient was not correlated with the sequencing depth (Supplementary Fig. 12).

Water samples from each site were collected near the bottom (<10 cm) and filtered in four Sterivex (0.22 μm) filters. Four independent replicates of seawater were prefiltered through nets of 100 μm and 20 μm by gravity. Two liters of water per replicate were filtered. Microbial cells retained in the filter received SET buffer and were stored at 30°C until DNA extraction. DNA extraction was performed using modified column purification protocol (Nucleospin Tissue, Macherey-Nagel, Dueren, Germany), as previously described (Bruce et al., 2012 (link)). A pool of DNA extracted from the filters was used for sequencing. Quality control was performed with Nanodrop absorbance and quantification by using the 2100 Bioanalyzer and Qubit High Sensitivity DNA Kit (Agilent, Santa Clara, CA, United States).