Plant total rna purification mini kit

Total RNA was extracted from flowers and leaves of R. kiyosumense using the Plant Total RNA Purification Mini Kit (Favorgen, Ping-Tung, Taiwan). The isolated total RNA was converted into cDNA with NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (New England BioLabs, Ipswich, MA, USA). A sequence library for isoform sequencing (Iso-Seq) was prepared using the Iso-Seq Express Template Preparation Kit (PacBio) and sequenced on the PacBio Sequel system (PacBio). Transcript isoforms for each sample were generated with the Iso-Seq3 pipeline (PacBio) implemented in SMRTlink (PacBio). The transcript sequences were aligned to the pseudomolecule sequences of R. ripense with Minmap2.

Leaf tissue or epidermal peels were harvested from 4‐ to 6‐week‐old plants and frozen in liquid nitrogen. RNA was extracted using a Plant total RNA purification mini kit (Favorgen^®). One microgram of RNA was used for cDNA synthesis using a MultiScribe Reverse Transcriptase (Applied Biosystems). PCR was performed on a 7500 thermocycler using SYBR‐green (both from Applied Biosystems). SlActin was used as endogenous control. Oligonucleotides used in this study are described in Table S1. Data analysis shown was done using three technical replicates from one biological sample; similar results were obtained with at least two additional independent biological replicates.

Total RNA was extracted from 14-d-old seedlings using the Plant Total RNA Purification Mini kit (Favorgen) according to the manufacturer’s protocol. RNA was then diluted to 10 ng/µl and reverse-transcribed into cDNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). qRT-PCR was performed using the TOPreal qPCR PreMIX kit (Enzynomics) in a 20 µl reaction on a Real-Time PCR LightCycler 480 System (Roche). Gene-specific primers were designed for AHA1 (forward: 5′-GGACAAGTTCGGTGTGAGGT3′, reverse: 5′-ACCAACTCCTTGACCTGGTG-3′) and AHA2 (forward: 5′-TGCTCAAAGGACACTTCACG-3′, reverse: 5′-GCCCTTTAGCTTCACGACTG-3′). Relative expression values were determined using UBQ10 (forward: 5′-GGCCTTGTATAATCCCTGATGAATAAG-3′, reverse: 5′-AAAGAGATAACAGGAACGGAAACATAGT-3′) as an internal reference and the comparative Ct method (2^-∆∆Ct). All qPCR analyses were performed in triplicate using three independent biological replicates.