Bioanalyzer 2100 rna nano 6000 reagent kit

Before beginning any of the library preparations, the RNA for each sample is quality checked using Agilent’s Bioanalyzer 2100 RNA Nano 6000 reagent kit. Each of the samples will ideally have an RNA Integrity Number (RIN) value higher than 8, indicating minimal sample degradation. We used Illumina’s TruSeq Total RNA Stranded Library Preparation Kit to achieve a range from 100 ng to 1 µg of total RNA. Generally, a concentration of 400–500 ng is used per sample, as that concentration falls in the middle of the indicated range. Library preparation is then performed according to the manufacturer’s instructions. Each sample is given a unique index to distinguish it from other samples. The final library size distribution is checked using a Bioanalyzer DNA High Sensitivity assay and if found to be within the correct size range, Qubit is used to determine the concentration of each sample. Samples are then pooled by concentration and an accurate concentration for the pool is determined using the Kapa Library Concentration Kit.

RNA quality for each sample assessed using Agilent's Bioanalyzer 2100 RNA Nano 6000 reagent kit, before library preparations. RNA integrity number value higher than 8, indicating minimal sample degradation, was required. Illumina TruSeq Total RNA Stranded Library Preparation Kit was used to generate a range of total RNA from 100 ng to 1 μg. Library preparation was performed according to the manufacturer's instructions. The final cDNA library size distribution was checked using a Bioanalyzer DNA High Sensitivity assay, followed by analysis Qubit to determine the concentration of each sample. Samples are then pooled and the accurate concentration for the pool is determined using the Kapa Library Concentration Kit.