Quant it dsdna high sensitivity assay kit

Genomic DNA was purified using a FastDNA Spin Kit for feces (MP Bio, Santa Ana, CA, U.S.A.) according to the manufacturer's instructions, except that the final elution was in 200 μL of DNAse-free water rather than 60 μL. Plasmids were purified with a NucleoSpin Plasmid Miniprep kit. The DNA was quantified using a Quant-iT dsDNA high sensitivity assay kit (Thermo Fisher, Waltham, MA, U.S.A.) and normalised to 0.2 ng.μL⁻¹ in 10 mM Tris-HCl. Sequencing libraries were prepared with the Nextera XT DNA Library Prep kit (Illumina, San Diego, CA, U.S.A.). Libraries were quantified using the Quant-iT dsDNA high sensitivity assay kit. Genome samples were pooled in equal quantities, while plasmid samples were first pooled together and the whole plasmid pool was added to the genomic pool at one-tenth the quantity of a genomic sample. The final pool was then run at a final concentration of 1.8 pM on an Illumina NextSeq 500 instrument using a mid-output sequencing kit for 150 bp paired-end reads. Sequences were quality checked by FastQC v0.11.7 and trimmed with Trimmomatic v0.36 with a minimum read length of 40 bp and a sliding window of 4 bp with average quality of 15. The reads were then mapped against the expected sequence for each sample in Geneious R11.1 with the Geneious mapper and default settings.

Genomic DNA was purified using a FastDNA Spin Kit for feces (MP Bio, Santa Ana, CA, U.S.A.) according to the manufacturer’s instructions, except that the final elution was in 200 μL of DNAse-free water rather than 60 μL. Plasmids were purified with a NucleoSpin Plasmid Miniprep kit (Macherey-Nagel, Düren, Germany). The DNA was quantified using a Quant-iT dsDNA high sensitivity assay kit (Thermo Fisher, Waltham, MA, U.S.A.) and normalised to 0.2 ng.μL^− 1 in 10 mM Tris-HCl. Sequencing libraries were prepared with the Nextera XT DNA Library Prep kit (Illumina, San Diego, CA, U.S.A.). Libraries were quantified using the Quant-iT dsDNA high sensitivity assay kit. Genome samples were pooled in equal quantities, while plasmid libraries were first pooled together and the whole plasmid pool was added to the genomic pool at one-tenth the quantity of a genomic sample. The final pool was then run at a concentration of 1.8 pM on an Illumina NextSeq 500 instrument using a mid-output sequencing kit for 150 bp paired-end reads. Sequences were quality checked by FastQC v0.11.7 and trimmed with Trimmomatic v0.36 with a minimum read length of 40 bp and a sliding window of 4 bp with average quality of 15. The reads were then mapped against the expected sequence for each sample in Geneious Prime 2019 with the Geneious mapper and default settings.

For cfDNA isolation, an automatic bead-based Maxwell RSC cfDNA Plasma kit (Promega, Fitchburg, WI, USA, AX1115) was used according to the manufacturer’s instructions. The choice of a kit was based on previous results by our group [12 (link)]. To quantify the concentration of total (double-stranded) DNA, a Quant-iT dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was employed according to the manufacturer’s instructions. For mutational cfDNA analysis of plasma and PJ samples, DNA libraries were prepared using 25 ng cfDNA input from PJ. From plasma samples, all available cfDNA (up to 25 ng) was used.
To prepare the tumor tissue samples for sequencing, the tissue was washed twice with PBS and subsequently treated with 550 µL lysis buffer and 20 µL protease overnight at 37–55 °C. RNase A (3 µL) was added and incubated for 15–60 min at 37 °C. Subsequently, a Chemagic MSM1 isolation robot (PerkinElmer Chemagen Technology, Baesweiler, Germany) was used to isolate the DNA according to the manufacturer’s recommendations.

Exosomal RNA was isolated using a commercially available total exosome RNA isolation kit (Qiagen’s exoRNeasy Serum Plasma Kit). Once exosomal RNA was isolated, it was further analyzed by sequencing. RNA quantity, 260/280 ratios, and 260/230 ratios, were assessed by Nanodrop (ThermoFisher). RNA integrity was verified on a Bioanalyzer 2100 using an Agilent RNA 6000 Nano Assay. Small RNA libraries were prepared according to the manufacturer’s protocol using a NEBNext® Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs). Libraries were size selected using a Pippen Prep (Sage Science), and quality-checked by Bioanalyzer using an Agilent High Sensitivity DNA Kit. Libraries were quantified by a Quant-iT™ dsDNA High Sensitivity Assay Kit (ThermoFisher) and sequenced on an Illumina NextSeq500 using a mid-output V2 kit.

mIMCD3 cells were seeded in 96-well plate with 1500 cells/well in duplicates, and were cultured in media containing normal (5.5 mM) or high (30 mM) glucose concentrations and were exposed to normoxia or hypoxia for 24 hr before analysis. Caspase 3/7 activity was evaluated using Caspase-Glo 3/7 assay kit (Promega) on the GloMax Luminometer (Promega) according to the manufacturer’s instructions. The caspase 3/7 activity was finally normalized to the DNA concentration in each well using Quant-iT dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific) measured using GloMax Discover Microplate Reader (Promega).

To resolve K. pneumoniae at the genus level, the V1-V3 region of the 16S rRNA gene was sequenced instead of the V4 region. V1–V3 sequencing libraries were generated by PCR using barcoded primers flanking V1 (27F, 5’-AGAGTTTGATCCTGGCTCAG-3’) and V3 (534R, 5’-ATTACCGCGGCTGCTGG-3’). PCR amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter), quantified using the Quant-iT dsDNA high-sensitivity assay kit (Thermo Fisher), pooled in equimolar amounts, and re-purified with the MinElute PCR purification kit (Qiagen). Sequencing was performed on an Illumina MiSeq at the NIH Intramural Sequencing Center (NISC).