GSM5426185: Arabidopsis thaliana; Arabidopsis thaliana; ncRNA-Seq
Total RNA samples were prepared using TRIzol® reagent or TRIzol LS® reagent for liquid samples (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer's recommendations. Contaminating DNA was degraded by DNase I (M0303S, New England Biolabs, MA, USA) treatment following manufacturer's instructions. Total RNA was shipped on dry ice and analyzed by Arraystar Inc. RNA sequencing service (Rockville, MD). The purity, quality, and concentration of total RNA samples were determined with NanoDrop ND-1000 (Thermo Fisher Scientific) and 2100 Bioanalyzer (Agilent, Santa Clara, CA). Total RNA of each sample was used to prepare the miRNA sequencing library, which included the following steps: (1) 3′-adapter ligation, (2) 5′-adapter ligation, (3) cDNA synthesis, (4) PCR amplification, and (5) size selection of approximately 120 to 150 bp of PCR-amplified fragments (corresponding to approximately 8 to 30 nt of small RNA). The complete libraries were quantified by Agilent 2100 Bioanalyser. Samples were diluted to a final concentration of 8 pM, denatured as single-stranded DNA, and cluster generation was performed on the Illumina cBot using TruSeq Rapid SR cluster kit (GD-402-4001, Illumina, San Diego, USA). Afterward, the clusters were sequenced for 51 cycles on Illumina HiSeq 2000 using TruSeq Rapid SBS Kits (FC-402-4002, Illumina, San Diego, USA), as per the manufacturer's instructions.