High sensitivity dsdna quanti it assay kit

MDSC-derived RNA samples were normalized, and a RNA input of 100 ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490 upstream and the NEXNext Multiplex Oligos for Illumina #E7600 downstream (New England Biolabs, Frankfurt am Main, Germany). Ampure XP beads (Beckman Coulter, Brea, CA, USA) were used for double-stranded cDNA purification. mRNA sequencing libraries were quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher Scientific) on a Synergy HTX (BioTek). Library molarity averaged at 134 nM. Final library size distribution was assessed (smear analysis of 364 bp average and adapter dimer presence <0.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 12-channel Fragment Analyzer (Agilent Technologies). All sequencing libraries passed quality check, were normalized, pooled, and spiked in with PhiX Control v3 (Illumina, San Diego, CA, USA). The library pool was subsequently clustered with the HiSeq 3000/4000 SR Cluster Kit on a cBot and sequenced on a HiSeq 3000 Sequencing System (Illumina) with single index, single read at 85 bp length (Read parameters: Rd1: 85, Rd2: 8), reaching an average depth of 29 million Pass-Filter reads per sample (11% CV).

Up to 1.6 ng of total RNA was used for cDNA synthesis, conducted with the SMART-Seq® v4 Ultra Low Input RNA kit (Takara Bio). Depending on the input amount, 12-15 amplification cycles were conducted. After clean-up, up to 10 ng of cDNA was used to generate the final sequencing libraries with the tagmentation-based DNA Prep Kit (#20018705) and the IDT® DNA/RNA UD Indexes Set A (#20026121), both Illumina®. The index PCR was conducted with 9 or 10 cycles depending on cDNA amount, while the final library was eluted in 30 µL EB Buffer. Low input mRNA libraries were then quantified using the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). Library molarity averaged at 21 nM. Libraries were also assessed for size distribution and adapter dimer presence (<0.5%) by the High Sensitivity NGS Fragment DNF-474 Kit on a 48-channel Fragment Analyser (Agilent). All sequencing libraries were then normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina). The library pools was subsequently clustered on an SP Flow Cell and sequenced on a NovaSeq 6000 Sequencing System (Illumina) with dual index, paired-end reads at 2 ×100 bp length (Read parameters: Rd1: 101, Rd2: 10, Rd3: 10, Rd4: 101), reaching an average depth of 26 million Pass-Filter reads per sample (18.2% CV).