GSM4647853: PolyA RNA INS Rep 2; Lemna gibba; RNA-Seq
Fronds grown on inorganic nitrogen or on organic nitrogen medium were harvested and immediately frozen in liquid nitrogen. For each condition three biological replicates were analyzed. Total RNA was extracted by grinding 200 mg frozen tissue to a fine powder in liquid nitrogen with a mortar and pestle, followed by extraction with TRIzol Reagent (Invitrogen) and ethanol precipitation. Total RNA was resuspended and treated with RQ1 RNase-free DNase (Promega) on an RNA Clean & Concentrator-5 column (Zymo). The purity and concentration of RNA were determined by a NanoDrop Nd-1000 spectrophotometer (Thermo Scientific) and a Qubit fluorometer (Qiagen). Polyadenylated RNA was enriched from total RNA with the Dynabeads mRNA Purification Kit (Life Technologies). Indexed, directional RNA-seq libraries were prepared with ScriptSeq v2 reagents (Epicentre) from 40-50 ng of polyA-selected RNA according to the manufacturer's instructions. Libraries were sequenced on an Illumina HiSeq 2000 instrument generating 101bp paired-end reads.