title: RNA-Seq of Phragmites australis: leaves
design: Tissue of three leaves was taken from each of three stems; a 0.5 cm2 piece of a second or third from the top fully developed and visually healthy leaf was cut in the middle position from the leaf base. Leaf material was pooled and homogenized in liquid nitrogen with mortar and pestle, then its total RNA was extracted using the innuPrep Plant RNA Kit (Analytik Jena, Jena, Germany) according to the manufacturers protocol. RNA was objected to DNase treatment using DNase I (Thermo Scientific, Waltham, MA, USA) according to the manufacturers protocol and objected to strand-specific cDNA library preparation using random hexamers. Library was sequenced on Illumina NovaSeq 6000 (Illumina Inc., San Diego, CA, USA) in S4 PE150 XP mode. Ribosomal RNA was filtered out from the raw reads using the program SortMeRNA v4.3.4 (Kopylova et al. 2012). Then the reads were trimmed for Illumina adapters and low-quality bases and the low-quality reads were discarded using trimmomatic v0.33 (Bolger et al. 2014) with the following parameters: ILLUMINACLIP:TruSeq3-PE.fa:2:30:10, SLIDINGWINDOW:5:30, MINLEN:100. Quality of the resulted reads were evaluated using programs fastQC v0.12.0 (Andrews 2010) and multiQC v1.14 (Ewels et al. 2016). Reads were taxonomically classified using the Kraken2 tool (Wood et al. 2019) and the KrakenTools suite (Lu et al. 2022). Sequence databases used for classification included Standard (covering archaea, bacteria, viruses, plasmids, human, and vectors) and PlusPFP-16 (including protozoa and fungi), both released on 09-04-2024.
library_name: c_Ro8x