title: GSM6746160: CT_R3; Salix viminalis; RNA-Seq
library_construction_protocol: Root samples (250 mg) were ground into a fine powder using mortar and pestle in liquid nitrogen. Total RNA was extracted following a modified CTAB buffer extraction protocol (Legay et al., 2015) and cleaned using the RNeasy plant mini kit (Qiagen, Leusden, The Netherlands) according to the manufacturer's instructions, using an extra DNAse I treatment to remove genomic DNA. RNA purity and quantity were assessed by measuring the absorbance at 230, 260 and 280 nm using a Nanodrop ND1000 spectrophotometer (Thermo Scientific, Villebon-Sur-Yvette, France). Total RNA integrity was assessed using the RNA Nano 6000 assay (Agilent Technologies, Diegem, Belgium) and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All RNAs had RIN above 7. Libraries were prepared using SMARTer Stranded RNA-Seq kit (Takara Bio Inc., Mountain View, CA, USA) following the manufacturer's instructions. Briefly, mRNA was isolated from 5 µg of total RNA using Magnosphere UltraPure mRNA Purification Kit (Takara Bio Inc.). The quality of the purified mRNA was assessed using RNA 6000 Pico assay (Agilent Technologies) and a 2100 Bioanalyzer. After purification, mRNA was sheared into fragments of approximately 300 bp, reverse-transcribed into cDNA using random primers, and ligated to Illumina Indexing Primer Set (Takara Bio Inc.), corresponding to Illumina TruSeq indexes 1 to 12. An enrichment step was carried out using 13 cycles of PCR. The samples' average fragment size was determined using a High Sensitivity DNA assay (Agilent Technologies) and a 2100 Bioanalyzer. The pooled libraries were sequenced on an Illumina NextSeq500 (Illumina NextSeq 500/550 High Output Kit v2.5 (300 cycles)) to generate 150 base-pairs pair-end reads.