RNA-seq of RAP1-deletion mutant 3 in low iron
The purified RNA was used to prepare the sequencing library with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. Briefly, mRNA was purified from total RNA (1 g) by oligo(dT)-coupled magnetic beads and fragmented into small pieces at elevated temperatures. First-strand cDNA was synthesized using reverse transcriptase and random primers. After generating double-strand cDNA and adenylation on 3 ends of DNA fragments, adaptors were ligated to the ends of the fragments. The resulting products were enriched through PCR and purified using the AMPure XP system (Beckman Coulter, Beverly, USA). The libraries were analyzed and qualified by a Qsep400 Bio-Fragment Analyzer (Bioptic Inc., New Taipei City, Taiwan) and a Qubit 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA). The qualified libraries were then sequenced on an Illumina NovaSeq platform with 150 bp paired-end reads generated by Genomics BioSci & Tech Co., New Taipei City, Taiwan.