PAT-seq Library Preparation for RNA-seq

Total RNA was extracted with a TaKaRa MiniBEST Plant RNA Extraction Kit and genomic DNA was removed by DNaseI (New England Biolabs). PAT-seq libraries were prepared as previously described with modifications (Lin et al., 2020 (link)). Two micrograms of total RNA were fragmented by 5× first strand buffer (TaKaRa) at 94°C for 4 min. Poly(A) RNAs were then enriched by oligo(dT)₂₅ beads (New England Biolabs). Reverse transcription was performed with oligo d(T)₁₈ primers by SMARTScribe™ Reverse Transcriptase (TaKaRa) for 2 h at 42°C. Then, a modified 5′ adaptor and SMARTScribe Reverse Transcriptase were added for another 2 h at 42°C. The cDNA generated was then purified with AMPure beads and amplified with Phire II (Thermo Fisher Scientific). The amplification products were then separated on a 2% agarose gel and 300–500 bp fragments were purified with a Zymoclean Gel DNA Recovery Kit. The concentration and quality of libraries were tested by a Qubit 2.0 and an Agilent Bioanalyzer 2100, and then sequenced on an Illumina HiSeq 2500 platform with 100-bp rapid sequencing mode.