2100 expert software

The FF sample was thawed and RNA was extracted using the Qiagen RNeasy kit [12 , 14 (link)]. For FFPE samples, RNA was extracted from adjacent tissue sections for each of three commonly-used commercial kits: N – Norgen (FFPE RNA purification Kit, Norgen, Thorold, Canada), Q – Qiagen (AllPrep DNA/RNA FFPE kit, Qiagen, Valencia, CA) and R – Roche (High Pure FFPE RNA Micro Kit, Roche, Indianapolis, IN). Two replicate RNA extractions were obtained per sample for each kit.
DNase I treatment was applied during both the FF and FFPE RNA isolation protocols. RNA concentration was quantified by Nanodrop (Nanodrop Technologies, Wilmington, DE). The RNA quality was analyzed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to produce an electrophoresis trace from which the RNA integrity number (RIN) and DV200 index were calculated using the 2100 Expert Software (Agilent Technologies). RIN is an algorithm used to estimate the integrity of RNA based on a combination of different features. RIN varies from 1 to 10, where 10 means perfect RNA integrity [15 (link)]. DV200 metric is the percentage of RNA fragments longer than 200 nucleotides and was found as a reliable determinant for RNA quality [16 (link)].

For RNA gel electrophoresis the Agilent RNA 6000 Nano Kit was used and the gel setting was prepared following the manual. Samples were run on a 2100 Bioanalyzer Instrument with the 2100 Expert Software (Agilent). Samples were applied in triplicates. As a negative control a particle suspension based sample was used that resulted in a “too low” signal in the Qubit® concentration measurement, to identify possible background noises that might have been generated by residual nanoparticles leftover in the samples after precipitation, like those of inorganic substrates or organic pigments.

To assess T7E1 and Surveyor nuclease digestion, we performed capillary electrophoresis on an Agilent 2100 Bioanalyzer. We used the DNA 1000 LabChip kit for electrophoresis of double-stranded DNA with detection by an intercalating dye and loaded 1 µL of the unpurified reaction products on the DNA chips. We used the 2100 Expert software (Agilent Technologies) for quantification and sizing of the digestion products. Data were exported in .csv files and electropherograms were drawn using R statistical software scripts.

Alternative splicing changes identified from RNA-seq were validated by RT-PCR using specifically designed primers (Supplementary Table 3). To confirm cassette exons, the primers were adjacent to the predicted splicing event. This approach allowed us to discriminate between variants based on their fragment sizes. For alternative first exon usage (AFE) validation, we have designed primers spanning regions that are unique to the isoform of interest (Fig. 6g), and then normalized the results by the housekeeping gene β-actin. cDNA was amplified using MyTaq Red DNA polymerase (Bioline, London, UK), and PCR products were analyzed using an Agilent 2100 Bioanalyzer system (Agilent Technologies, Wokingham, U.K.). The molarity of each PCR band corresponding to a specific splice variant was quantified using the 2100 Expert Software (Agilent Technologies, Diegem, Belgium), and used to calculate the ratio inclusion/exclusion (SE) or isoform-X/β-actin (AFE).

To evaluate the size distribution of cfDNA fragments, samples were assessed using the Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and the DNA HS kit (Agilent Technologies, Santa Clara, CA, USA, 5067-4626 & 5067-4627). Each sample was compared with two size-standardized internal controls (of 35 bp and 10,380 bp) and a DNA ladder (15 peaks). The profile of fragment sizes was generated using the 2100 Expert Software (Agilent Technologies, Santa Clara, CA, USA).
To determine the concentration of cfDNA, two blinded independent complementary assays were carried out. First, the quantification of double-strand DNA was assessed using a Qubit 2.0 Fluorometer and the Qubit dsDNA HS Assay Kit (Life Technologies, Q32854, Carlsbad, CA, USA) according to the manufacturer's instructions. The concentration was expressed in ng/mL and then converted to ng/μL. We also quantified amplifiable cfDNA by quantitative PCR (qPCR) using the Quantifiler Human DNA Quantification Kit (Applied Biosystems, PN4344790F, Foster City, CA, USA) according to the manufacturer's instructions, with the hTert gene (human telomerase reverse transcriptase). Based on the C_T value of the internal positive control (IPC), we determined both the number of copies of gene/mL of plasma or ng/μL of cfDNA of our samples.

To overcome the problem of a low amount of DNA obtained from a single offspring, ecDNA samples from all offspring of one mother were pooled and concentrated using Concentrator plus (Eppendorf, Hamburg, Germany). Pooled samples were analyzed employing the 2100 Bioanalyzer Instrument using the Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, California, USA) according to the manufacturer’s instructions. Briefly, 1 μL of each sample was used for on-chip electrophoresis and run for 45 min. A DNA standard ladder was used. The fragmentation profile is shown using a gel-like image generated by 2100 Expert Software (Agilent Technologies, Santa Clara, California, USA).

RNA integrity was measured for RNA extracted from the 80 FFPE melanoma samples of the main study cohort and from the 13 cell lines using the RNA 6000 Nano Kit and a 2100 Bioanalyzer (Agilent Technologies, Glostrup, Denmark) according to manufacturer’s instructions. Results were evaluated using the 2100 Expert software (Agilent Technologies). RNA Integrity Numbers (RIN values) are displayed in Table of Supplemental Digital Content 1, http://links.lww.com/MR/A190.