GSM3272544: PTA_rep1; Populus trichocarpa; ncRNA-Seq
Total RNA was extracted from the root system of three of the eight replicates per genotype per treatment by combining a CTAB extraction method and the Spectrum™ Total Plant RNA extraction kit (Sigma) as reported in Bryan et al. (2016) Prior to synthesizing sequencing libraries, RNA templates were quantified using a Nanodrop Spectrophotometer (Thermo Scientific) and RNA quality was determined using BioRad Experion™. Samples with a 260/280 reading between 2.0 and 2.2 and a RIN >7 were determined to be of high enough quality and the high-quality RNA samples were used for sRNA-sequencing library preparation with an Illumina TruSeq small RNA sample prep kit (Illumina, CA, USA). As specified in the Illumina protocol, small RNA fragments were selected after PCR amplification by gel purification to capture RNA approximately 20bp-170bp in length.  The Illumina TruSeq small RNA library prep workflow is considered to be stranded because adapters are ligated directionally, facilitating maintenance of strand information during analysis.