RNA-seq Library Preparation and Sequencing

To ensure appropriate RNA quality and evaluate RNA degradation, total RNA was analyzed using Agilent Bioanalyzer 2100 (Agilent Technologies, USA) and RNA 6000 Nano Kit (Agilent). An average RNA integrity number (RIN) of 8 was obtained. Total RNA was used for Illumina library preparation and RNA-seq [60 (link)]. 2.5 µg total RNA was used for indexed library preparation using Illumina’s TruSeq™ RNA Sample Prep Kit v2 following the manufacturer’s instruction. Libraries were pooled and sequenced (five samples per lane) using a HiSeq 2000 (Illumina) in single read mode with 50 cycles using sequencing chemistry v3. Sequencing resulted in approximately 40 million reads with a length of 50 bp (base pairs) per sample. Reads were extracted in FastQ format using CASAVA v1.8.2 or v1.8.3 (Illumina).