title: GSM9600478: Lemna minor, NodeTract region, replicate 4; Lemna minor; RNA-Seq
library_construction_protocol: Duckweed samples: Samples were recovered from RNAlater by sieving through 70uM cell strainers, blotted from underneath with tissue paper to remove excess RNAlater, and snap frozen. Samples were ground on liquid nitrogen in 1.5ml Eppendorf tubes with plastic pestles. RNA was extracted using a RNeasy UCP Micro Kit (Qiagen) as per manufacturer's instructions. Taro leaf-derived tissue (Apical, Basal regions): Tissue was ground to fine powder using a mortar and pestle on liquid nitrogen. 3 replicate aliquots of approximately 100mg were taken, added to 450ul buffer RLT and snap frozen. RNA extraction was performed on a single aliquot using the RNeasy Mini Kit (Qiagen) as per manufacturer's instructions with an additional RPE wash step. Taro sheath and petiole tissue: Tissue was ground to fine powder using a mortar and pestle on liquid nitrogen. 3 replicate aliquots of approximately 100mg were taken, added to 450ul buffer RLT and snap frozen. All three aliquots were extracted using the RNeasy Mini Kit (Qiagen), then pooled and cleaned up using the RNeasy PowerClean Pro Cleanup Kit as per manufacturer's instructions. RNA integrity was assessed using the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 (Agilent Technologies). Library preparation and sequencing were performed by Novogene. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. After adenylation of 3' ends, adaptors with hairpin loop structure were ligated. Library fragments of 370-420 bp were purified with AMPure XP system (Beckman Coulter). PCR was performed with Phusion High-Fidelity DNA polymerase. Clustering was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Libraries were sequenced on an Illumina NovaSeq platform generating 150 bp paired-end reads.