RNA-Seq Library Preparation Workflow

Nuclear RNA isolated using the TRIzol method (Invitrogen) was further purified and DNase-treated using the Monarch RNA Cleanup Kit (New England Biolabs). Library preparation and RNA-seq were performed at the Genomics Core Facility (University of Regensburg, www.kfb-regensburg.de), employing the following modules: NuGEN Universal Plus RNA-Seq with NuQuant User Guide v3 (Tecan Genomics) in combination with Arabidopsis rRNA AnyDeplete module, the Illumina NextSeq 2000 System (Illumina), and the KAPA Library Quantification Kit-Illumina/ABI Prism (Roche Sequencing Solutions). To judge final library complexities (vs. PCR duplicates) unique molecular tags were used (Smith et al., 2017 (link)). Equimolar amounts of each library were sequenced on an Illumina NextSeq 2000 instrument controlled by the NextSeq 2000 Control Software (NCS, v1.4.0.39521), using 50 cycles P3 Flow Cell with the dual index, paired-end run parameters. Image analysis and base calling were done by the Real Time Analysis Software (RTA, v3.9.2). The resulting ‘.cbcl’ files were converted into ‘.fastq’ files with the bcl2fastq (v2.20) software.