Qubit dna hs assay kit

DNA and RNA isolations were performed from both FFPE and frozen sections using FFPE DNA Purification Kit (Cat. 47400, Norgen Biotek Corp, Thorold, Canada) and RNA/DNA Purification Kit (Cat. 48700, Norgen Biotek Corp.) respectively, according to the manufacturers protocol. Nucleic acids were quantified with Qubit 2.0 fluorometer using Qubit RNA HS assay kit and Qubit DNA HS assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The assessment of nucleic acids integrity (DIN and RIN) was performed with Agilent 4150 TapeStation System (Agilent Technologies, Santa Clara, CA, USA).

RNA was processed for library preparation using the TruSeq^® RNA Access Library Preparation Kit (Illumina, San Diego, CA, USA) that allows generating libraries starting from degraded and low yield RNA. Briefly, the first and second cDNA strands were synthetized from input RNA in order to be adaptor-tagged, labeled and amplified. CDNA from each sample was then pooled. Pooling strategy was decided considering tissues and subjects uniformity. Each pool contained two to four samples of the same tissue and included both patients and controls. Pooled samples were enriched by a double step of probes hybridization. The enriched targets were captured by streptavidin labelled beads, cleaned up and amplified to obtain the final multiplexed libraries. Libraries quality was checked for fragments distribution using Agilent DNA High Sensitivity Kit on an Agilent 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, USA) and quantified by Qubit^® DNA HS Assay kit on a Qubit^® 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The libraries were then sequenced on an Illumina HiSeq^® 2500 platform (Illumina^®) with a rapid paired-end sequencing (2 × 101 bp). The protocol for libraries generation was executed step by step except for the adjustment of cDNA amount used for hybridization (details shown in the results section).

Additional rRNA removal step was performed on n = 6 DNase treated RNA (two for each kit). The rRNA removal was performed using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs, Ipswich, MA) as per the manufacturer’s instructions. Each viral extract was then subjected to Whole Transcriptome Amplification (WTA) using the QIAseq FX Single Cell RNA Library Kit (Qiagen, Hilden, Germany) as per manufacturer’s instructions to generate the cDNA. Amplified cDNA was quantified using the Qubit® DNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA) with the Qubit® 3.0 Fluorometer.

E. cecorum sequencing libraries were prepared using a Nextera™ XT DNA Sample Preparation Kit and a Nextera™ XT Index Kit (Illumina Inc., San Diego, CA, USA). Illumina libraries were then quantified using a Qubit^® DNA HS Assay Kit in a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and the size of the fragment libraries was checked using an Agilent 2100 Bioanalyzer System with an Agilent HS DNA Kit (Agilent Technologies, Santa Clara, CA, USA). Illumina libraries were sequenced on an Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) using a MiSeq v2 reagent kit with 500 cycles and a paired-end read length of 2 × 250 bp. Resulting paired-end sequencing reads were de novo assembled into contigs using A5-miseq assembler [27 (link)] and contigs were annotated using Prokka [28 (link)]. Prophage analysis was done using PHASTER [29 (link)]. The list of the web-based and command line tools used in our study is provided in Table S1.

After 8 days since the transfer of germinated seeds into Hoagland solutions, fresh leaves were collected from each pot and processed to extract DNA. For each leaf sample, 50 mg was homogenized with 300 µL of RLT buffer in two ml Eppendorf tubes using a Tissue Lyser (Qiagen, Hilden, Germany) for 5 min at 30 hertz. The tubes were centrifuged at 6000 rpm for 3 min, and the supernatant obtained was processed using Biosprint 96 (Qiagen, Hilden, Germany) to extract DNA. The automatic extraction involved the use of six 96 well S-Block plates set up as follows. One plate filled with 300 µL of sample supernatant, 200 µL of isopropanol, and 20 µL of MagAttract magnetic beads (Qiagen, Hilden, Germany). A second plate with 500 µL of buffer RLT. The third and fourth plates with 500 µL of 96% ethanol. The fifth plate with 500 µL of 0.02% Tween solution and the last plate filled with 100 µL of nuclease-free water. After the extraction process, DNA was quantified with a Qubit fluorimeter (Thermo Fisher Scientific Inc., Waltham, MA, USA) using the Qubit™ DNA HS Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).

cfTNA was extracted from 4 to 8 mL of plasma using the MagMAX Cell-Free Total Nucleic Acid Isolation kit (Thermo Fisher Scientific, Waltham, MA, USA) or NextPrep-Mag cfDNA Automated Isolation Kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s protocol. Genomic DNA from buffy coat was extracted using the FlexiGene DNA Kit (Qiagen, Venlo, The Netherlands) or chemagic DNA Blood 400 Kit H96 (PerkinElmer, Waltham, MA, USA).
Genomic DNA of tumor tissue (both tumor and normal, if normal buffy coat DNA was absent) was extracted from ten 5 μm slices of formalin-fixed paraffin-embedded (FFPE) slides, which were macrodissected to leave only the tumor tissue, using GeneRead DNA FFPE Kit (Qiagen, Venlo, The Netherlands), according to the manufacturer’s protocol. The extracted cfTNA and genomic DNA were quantified using the Qubit DNA HS Assay Kit and Qubit DNA BR assay kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively. The quality and size of extracted cfTNA were evaluated using the High Sensitivity D5000 ScreenTape (Agilent, Santa Clara, CA, USA), whereas the quality of genomic DNA was evaluated using the Genomic DNA ScreenTape (Agilent, Santa Clara, CA, USA) with TapeStation System (Agilent, Santa Clara, CA, USA).

Pituitary tumor samples (10–50 mg) were powdered under liquid nitrogen. DNA was extracted and purified following proteinase K digestion using a TIANamp Genomic DNA Kit (TIANGEN Biotech, Catalog No. DP304-03). DNA concentrations were determined using a Qubit DNA HS Assay Kit (Thermo Fisher, Catalog No. Q32854). The DNA quality was assessed with Agilent 2,200 TapeStation Genomic DNA Analysis (Agilent Technologies).
Total RNA was extracted using TRIzol reagent (Thermo Fisher, Catalog No. 15596018). The RNA quality was checked by Agilent 2,200 TapeStation RNA Analysis (Agilent Technologies) to determine the RNA integrity number (RIN). The RNA was considered acceptable for cDNA library construction when the RIN was >7.0.