Rice experiment 3, mock+mock treated, replicate 2
Plant material was ground using mortar and pestle cooled by liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturers protocol. RNA was treated with DNase I (30 min at 37C) and subsequently purified by sodium acetate/ethanol precipitation. Quality and concentration of the input total RNA was checked with 'Quant-it ribogreen RNA assay' (Life Technologies) and with the DNF-472 HS RNA (15 nt) kit on the Fragment Analyzer (Agilent Technologies), respectively. The RIN values were 7.20  0.40 (average  standard deviation). Subsequently, 170 ng of RNA was used to perform an Illumina sequencing library preparation using the QuantSeq 3' mRNA-Seq Library Prep FWD Kit (Lexogen) including Unique Molecular Identifiers (UMI) according to the manufacturer's protocol using 15 enrichment PCR cycles. Libraries were quantified by qPCR, according to Illumina's protocol 'Sequencing Library qPCR Quantification protocol guide'. With a DNF-474 HS NGS Fragment (1-6000 bp) kit on Fragment Analyzer (Agilent Technologies) the library's size distribution and quality were evaluated. Libraries were equimolarly pooled and sequenced on an Element Biosciences Aviti sequencer together with 5% PhiX Sequencing Control library, generating 100 bp single-end reads.