Waus_spRNA-Seq_I3
Single-plant Wolffia RNA was extracted using the protocol of RNeasy plant mini kit (Qiagen cat# 74903). RNA concentration was measured using a Qubit RNA assay kit (ThermoFisher, Ref# Q32855A) in a Qubit 2.0 fluorometer (Life Technologies, Ref# REQ32866). Total RNA of 100 ng per sample was used as input material for RNA sample preparations. Libraries were generated using a NEBNext UltraTM RNA library prep kit Illumina (New England Biolabs, Ipswich, MA, USA, Cat# E7530) and index codes were added to attribute sequences to each sample. The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (New England Biolabs, Ipswich, Massachusetts, USA, Cat# E7490). First-strand cDNAs were synthesized by the NEBNext RNA First Strand Synthesis Module (New England Biolabs, Ipswich, MA, USA, Cat# E7525L). Second-strand synthesis was performed by the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, Ipswich, MA, USA, Cat# E6111L). Library was prepared using KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, Massachusetts, USA, Cat# KK8504). Products were purified with AMPure XP system (Beckman Coulter, Cat# A63882), and library quality was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, Ref# G2939BA).