Random PCR and SPIA Amplification for RNA-Seq

The DNA was amplified using random PCR as described elsewhere [14 (link)]. The PCR tag-sequence was cleaved away using EcoRV (Thermo Fisher, Waltham, MA, USA). The RNA was reverse-transcribed into cDNA and amplified by single-primer isothermal amplification (SPIA) using the Ovation RNA-Seq V2 kit (NuGEN, San Carlos, CA, USA) according to the manufacturer’s instructions. The random PCR products and the SPIA products were then purified using the GeneJET PCR purification kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. The concentrations of the purified random PCR products ranged from 18 to 25 ng/µL, and those of the purified SPIA products ranged from 36 to 66 ng/µL. All the products were sequenced at SciLifeLab/Genome Center (Uppsala, Sweden) using the Ion S5 XL system (Thermo Fisher, Waltham, MA, USA) and two 530 chips. The main type of sequencing errors associated with this platform are insertions and deletions (indels).