Dnf 488 high sensitivity genomic dna analysis kit

Longissimus thoracis skeletal muscle samples were collected at slaughter, stored in liquid nitrogen, and kept in a freezer at − 80 °C until processing. DNA extraction was carried out using the DNeasy® Blood & Tissue kit (Qiagen Inc., Germantown, MD, USA), according to the manufacturer's protocol. DNA concentration was measured using Qubit dsDNA High Sensitivity Assay (Thermo Fisher Scientific Waltham, MS, EUA) and the quality measured by Fragment Analyzer™ and the DNF-487 Standard Sensitivity or the DNF-488 High Sensitivity genomic DNA Analysis Kit (Advanced Analytical).

The size, purity and the molar concentration (nmol/l) of each amplicon library was measured using a Fragment Analyzer™ (Advanced Analytical Technologies, Inc., USA) and a DNF-910 dsDNA Reagent Kit (35 bp–1,500 bp). An equimolar pool (2 ng/μl/library) was prepared from the amplicon libraries using the Biomek 4000 Laboratory Automation Workstation (Beckman Coulter, USA). Agencourt AMPure XP (Beckman Coulter, USA) was used for removal of unincorporated primers and nucleotides using the manufacturer’s instructions (Agencourt AMPure XP v. B37419AA). The total concentration of the purified pooled amplicon library stock and three serial dilutions (undiluted, 1/5, 1/10) were analyzed using the Fragment Analyzer™ (Advanced Analytical Technologies, Inc., USA) and DNF-488 High Sensitivity Genomic DNA Analysis Kit in order to identify the optimum concentration range for the template preparation.

Genomic DNA was extracted with Allprep DNA/RNA mini kit (Qiagen). and concentrations measured using the Qubit® dsDNA BR Assay Kit (Thermo Fisher Scientific), followed by DNA quality assessment with the Fragment Analyzer^TM and either the DNF-487 Standard Sensitivity or the DNF-488 High sensitivity genomic DNA Analysis Kit (Advanced Analytical). RRBS libraries were prepared using the Premium Reduced Representation Bisulfite Sequencing (RRBS) Kit (Diagenode Cat# C02030033), according to the manufacturer’s protocol. 100 ng of genomic DNA were used to start library preparation for each sample. Following library preparation, samples were pooled together either by 8 (mouse contamination up to 10%), 7 (mouse contamination up to 25%), 5 (mouse contamination up to 40%), or 4 (mouse contamination up to 55%). In total, 16 pools were prepared. PCR clean-up after the final library amplification was performed using a 1.5× beads:sample ratio of Agencourt® AMPure® XP (Beckman Coulter). RRBS library pools quality control was performed by measuring DNA concentration of the pools using the Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific), and the profile of the pools was checked using the High Sensitivity DNA chip for 2100 Bioanalyzer (Agilent). Each RRBS library pool was deep sequenced on one lane HiSeq3000 (Illumina) using 50 bp single-read sequencing (SR50).