Nebnext globin rrna depletion kit human mouse rat

The mRNA expression in WT was determined on RNA isolated from FFT samples of WT and HK tissue, which were used for miRNA determination. Samples for mRNA expression were prepared using the NEBNext Globin&rRNA Depletion Kit Human/Mouse/Rat (New England Biolabs) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). mRNA expression profiles were determined after mRNA sequencing with 100 million reads per sample using a NovaSeq 6000 system (Illumina).

The RNA integrity number was measured with an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA) and an Agilent RNA 6000 Nano Kit (Agilent Technologies Inc.), as shown in Table 1. Next, 200 ng total RNA from each sample was subjected to RNA-seq library preparation. Initially, globin mRNA and ribosomal RNA (rRNA) were removed using the NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs, Ipswich, MA, USA) and NEBNext Multiplex Oligos for Illumina (New England Biolabs). An RNA-seq library of each sample was prepared by using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Indianapolis, IN, USA) according to the manufacturer’s protocol. The quality of the average size (340–380 bp) was validated by using an Agilent 2100 Bioanalyzer and the Agilent DNA1000 kit (Agilent Technologies Inc.). The amount was determined using the qPCR method with the Kapa Library Quantification Kit (Illumina). The sequencing was done using the MiSeq Reagent kit V3 on a MiSeq system (Illumina) based on pair-end reads (75 bp) according to the manufacturer’s instructions. Sequencing was performed by running 150 cycles.