Check Library

This step checks the concentration and quality of the final libraries.

1. Analyze the library using one of the following methods:
• If using the NextSeq 1000/2000 Sequencing System, dilute 1 μl library 1:10,000 dilution, and then analyze library using KAPA qPCR library quantification kit.
• For all other sequencing systems, analyze 1 µl library using one of the following:
• Agilent 2100 Bioanalyzer and DNA 1000 Kit
• TapeStation System and D1000 ScreenTape

The following figures provide example traces of final libraries generated from 25 ng and 100 ng Universal Human Reference (UHR) RNA input.

Bioanalyzer trace with 25 ng input

Bioanalyzer trace with 100 ng input

TapeStation trace with 25 ng input

2. [Optional] Analyze 2 µl library using the Qubit dsDNA BR Assay Kit for further quantification.

For intact RNA samples, the average fragment length is ~300–400 bp. The expected insert size is ~160 bp.