Illumina-Based RNA Sequencing Pipeline

RNA was extracted using the TaKaRa kit and cDNA libraries were made for RNA. From total RNA, mRNA was filtered using the Oligotex mRNA Midi Kit (QIAGEN, GERMANY), followed by quality and quantity check using a Nano-Drop 2000 spectrophotometer (Thermo Scientific, USA). The cDNA libraries were made using the Illumina manufacturing protocol (Ahmad et al., 2021a (link)). The total mRNA was subjected to first and second strand cDNA synthesis and adapter ligation, and low cycle enrichment was achieved by the TruSeq^®RNA HT Sample Prep Kit from Illumina (USA). The products were evaluated with the Agilent 2200 TapeStation and Qubit^®2.0 of Life Technologies (USA). Dilutions were made to 10 pM for generating in situ clusters on HiSeq2500 pair-end flow cell. The 2 × 100 bp sequencing was performed, resulting in 60 M reads per sample. Transcriptomic de novo was done with the Trinity program using default parameters (Grabherr et al., 2011 (link)).